1BioAP 3160 - LECTURES 1-5 January 23-February 1,

2012
Different cell organelles : scheme of a typical cell - How to recognie their ultrastructural
appearance - composition !ios"nt#esis an$ f%nction&s' of organelles an! of their
components: wor"ing our way out from the nucleus#
T(E )UCLEUS
$n most cells the most prominent organelle - Repositor" of genetic information -most cells
ha%e nucleus &e'ceptions : erythrocytes (they !o in bir!s )*, platelets+
- Disting%is#ing feat%re !et*een procar"otes an$ e%car"otes
- ,he n%clear en+elope, separates nucleoplasm - cytoplasm
-nuclear en%elope is a !ouble membrane
-has an inner membrane an! outer membrane
-inner membrane functions are relate! to nuclear f'n
-outer membrane is continuous with the cisternae of the ./
- )%clear pores allow e'change of materials (information* between nucleus an! cytoplasm -
0hy is separation nee!e! between nuclear material an! cytoplasm1
-nee! separation so that transcription an! translation are segregate!
0hat are the main physiological implications1
-allow the many proteins in%ol%e! in replication an! transc# to be concentrate! where they are
nee!e!, "eeps nuclear enymes separate from cytosolic enymes,
- the n%cleol%s, (2ppears !ar" in .3 because of concentration of ribosomes an! r/42 being
ma!e*, the regions of con!ense! (#eteroc#romatin* an! !isperse! (e%c#romatin* chromatin#
- +ariation in t#e amo%nt of n%clear material-cell- 3ultinuclearity, polyploi!ity, politeny, gene
amplification
- 5ariability in shape of the nuclei - often has physiologic significance
- 6ifferences in the relati%e amounts of heterochromatin7euchromatin8
Constit%ti+e %s fac%ltati+e #eteroc#romatin 9 const# hetero is always there, li"e centromeres,
telomeres, etc# Facultati%e is li"e maternal7paternal imprinting
- C-+al%e para$o. : amount of 6427haploi! nucleus !oes not correlate with organism
comple'ity - $t is now assume! that in mammals only about 1: of the 642 is in%ol%e! !irectly
in /42 synthesis (classic genes*
C#romatin / D)A 0 associate$ proteins ; most of content of nucleus
1
- minimal linear chromosome elements: D)A replication origin (const# hetero# <e=uences that
!on>t correspon! to acti%e genes but can bin! to components of replication apparatus*,
centromere (const# hetero# 0here mitotic spin!le attaches*, 1 telomeres (repetiti%e se=uences
necessary to pre%ent shortening of chromosome after replication*
- 1 molecule !ouble stran!e! 642 per chromosome (2?02@< .A$<,, e%en !uring interphase*
- in humans ma' length: 10 cm (2-3'10
B
bp*
- 642-bin!ing proteins in eu"aryotes: #istones - non-#istone c#romosomal proteins
(istones: about C0: of protein in the nucleus, total mass of histones about e=ual to that of 642
- highly conser%e!, relati%ely small, high proportion of positi%ely-charge! 22s (?ys, 2rg*
- 5 types of histones: nucleosomal histones (H22, H2D, H3, HE*, H1 histones
- H3 - HE among the most highly conser%e! proteins
- H1 less conser%e!, %arious types in each nucleus - Fooperati%e bin!ing
- Histones pac"age 642 - also contribute to gene regulation
- -2ll histones> core regions %ery similar, !ifferences at F an! 4 termini
- 3o!ifications : H1 phosphorylation (serine* 9contributes to con!ensation
H3,HE acetylation (lysine* - less con!ense! chromatin (642se $ sensiti%ity*
3ethylation 9mo!ification controls con!ensation an! e'pression
- methylation of H3 causes hetero# 6e%elopment an! gene silencing
Gbi=uitin-H22, only in interphase, acti%e genes
- 2ll of the abo%e possible because the octameric proteins all ha%e tails that stic" out of
octamer that get mo!ifie!
)on (istone Proteins,
-compose about 173 less than histones of total protein in cell
- !efine! as proteins lefto%er when histones ha%e been remo%e! from cell
- %ery heterogeneous
- less basic than histones
- ,hree possible functions: 1* structural components of chromatin in%ol%e! with higher or!er
compaction of nucleosomes, 2* enymes in%ol%e! with 642 replication, /42 transcription,
histone mo!ification, /42 processing 3* control of gene e'pression with specific recognition sites
on 642 9 inc finger
Se2%ence-specific D)A-!in$ing proteins, 642 fol!ing, initiation of 642 replication, control of
gene transcription8 Din! to 642 without !isturbing the !ouble heli'
- most gene-regulatory proteins bin! to the 642 maHor groo%e
2
- heli'-turn-heli' motif common feature of 642-bin!ing proteins (e'ample, homeo!omain
proteins*
- inc fingers (inc forms comple' with Fys an! His an! creates fingers that fit into maHor
groo%es*, leucine ipper, heli'-loop-heli'
- Fooperati%e bin!ing
- 3t#er non-(istone c#romosomal proteins, .nymes, pac"aging proteins, transcription
factors
STRUCTURE 34 C(R35AT6) : in interphase little can be seen by con%entional ,.3
)%cleosome mo$el : -compaction an! con!ensation of 642 to fit into nucleus ; nucleosome
- one nucleosome is lin"er 642, histone core, an! 1EC bp aroun! core
- basic informations lea!ing to it: 3iller sprea!s (floating chromatin on air7water interphase,
bea!s on a string appearance (11 nm !iameter*8 if you use pure 642, Hust get string an! no
bea!s -II bea!s must be proteins ; histones
3iller sprea!s on saline : 30 nm nucleoprotein fiber 9 this !emonstrate! the first le%el of
organiation, but still nee! two more to fit 642 in nucleus -II 3 stages of 642 pac"aging
- e'tensi%e pac"aging re=uire! to con!ense 5 cm 642 molecules into nuclei 5 Jm in !iameter
- 3icrococcal 642se !igestion : at low concentration 642 fragments of about 200 bp - at higher
concentration, 1EC bp fragments:
- smallest pieces of !igeste! 642 on gel electrophoresis were 205 bp, tells us that there are
pieces of 642 205 bp long that cannot be !igeste! 9 642 wrappe! aroun! histone core is
protecte! from !igestion#
- if you a!! higher conc# Kf 642ase, ta"e out the histone core an! the lin"er 642, get
fragment 1EC bp long# <o, lin"er region ; about 5Cbp
- nucleosome bea!s pro!uce! by !igestion with micrococcal nuclease
)%cleosome mo$el: octamer of H22,H2D,H3,HE - 1EC bp 642 wrappe! on it
- Histone H1 4K, part of octamer, it bin!s to the outsi!e of the core, "in! of seals the coil of
642 aroun! the octamer core#
- lin"er 642 (0-B0 nucleoti!e pairs*
- nucleosomes not ran!omly positione!: propensity of 642 heli' to form tight loops (se=uence-
!epen!ent*8
642-bin!ing proteins (64ase $, nuclease-hypersensiti%e sites*
- (istone (1 (si' closely relate! %arieties* pac"ages nucleosomes into the 30 nm fiber8 higher
or!er structures8 cooperati%e bin!ing# /egulate! by phosphorylation# 0hen phosphorylate!
3
chromatin becomes more compacte!#
- 11 nm fiber is lin"er, bea!, lin"er, bea!
- 30 nm fiber is bea!s on a string wrappe! aroun! itself: still !on>t "now its structure for sure
- R)A s"nt#esis in the presence of histones
C(R353S35ES : %isible !uring mitosis - number - shape characteristic for each species (23 in
humans* 9present all the time (!uh*
- Unitem" : 1 molecule of !ouble stran!e! 6427chromosome
- 2t least two more or!ers of compaction than in nucleoprotein (30 nm* fibers
- Highly con!ense! mitotic chromosomes
- e'amples %isible un!er the microscope: lampbrush chromosomes (amphibian oocytes,
meiotically-paire! chromosomes co%ere! with /42-protein comple'es* - polytene
chromosomes (6rosophila sali%ary cells, 102E stran!s of i!entical chromatin, light - !ar"
ban!s8 chromosome puffs*
- transcriptionally-acti%e chromatin is less con!ense!
- nuclear matri' may help organie chromosomes7chromatin in interphase nuclei
- Fon!ensation of chromatin in mitotic chromosomes (phosphorylation of histone H1, sister
chromati!s hel! together at centromeres8 transcriptionally inacti%e*
- chromosome ban!s (L ban!s;2-, rich8 / ban!s;L-F rich*
- in or!er to !econ!ense nucleosomes an! access 642 for replication, transc# an! gene
e'pression, use remo!eling comple' 2: starts unwin!ing process an! allows 642 bin!ing (non
histone* proteins to bin! an! hol! 642 open, then remo!eling comple' D !issociates the 642
bin!ing proteins an! the nucleosomes are allowe! to reform#
- !uring this process scaffol! proteins remain boun! to 642 ("eep it straightene! out*
(7-TE5 +ie* of c#romosomes : ?oope! !omains in chromosome sprea!s8 $n interphase,
chromatin of each chromosome !isperse! in the nucleus, an! intermingle!, an! 3GFH ?.<<
FK46.4<.6
Ban$s in c#romosomes - regions of !ifferent composition, co%ering much more than a single
gene
- ban!s are 4K, in!i%i!ual genes)
- Dan!s tell us that some regions of a chr# are more or less tightly woun!
- FH/K3K<K3.< 4K, G4$FK/3 6K04 ,H.$/ ?.4L,H
- Dan!ing helps us match chromosomes !uring "aryotyping
E
D)A REPL6CAT63)
- higher eu"aryotes always ha%e I1 replication origin
- ta"es place !uring the < phase of the cell cycle
- T#e replication apparat%s : stu!ie! mostly in bacteria (mutants* but assume! to be
essentially the same in eu"aryotes (howe%er, histones are present in this case )*# 2ccurate,
rapi! synthesis of 642 (500 nucleoti!es7sec in bacteria, 507sec in mammals*
- D)A stran$s f%nction as templates - replicating for"8 continuous synthesis in the 5M-3M
!irection (more accurate synthesis - proofrea!ing - tautomeric forms of bases*
Accepte$ mo$el of D)A replication
- 642 replication for" is asymmetrical (lea!ing stran!, lagging stran!, K"aa"i fragments*
- 1 642 polymerase in bacteria, at least 2 in eucaryotes:
- Nolymerase alpha has primase subunit that synthesies primers# 2lpha is too slow to
synthesie by itself so switch to polymerase !elta, much faster enyme# For lea!ing stran! this
switch only happens once# Happens more on lagging (switching from primers much more often*
- $n or!er to switch from alpha to !elta nee! NF42 (proliferati%e cell nuclear antigen*# $t>s also a
target for protein "inase-inhibitors 9 slows7stops 642 rep to allow repair to occur
- /NF (replisome progression comple'* bin!s to 642 on lagging stran!, an! NF42 bin!s to
/FF, an! NF42 helps remo%e pol-alpha an! replace with pol-O (switching*8
- accessory proteins: /N2 replication protein 2 ; <<D protein that stabilies the template stran!
- /FF replication factor F; stabilies the lagging stran! while NF42 bin!s an! switches alpha
out for !elta
- Nroofrea!ing mechanisms: tautomeric forms of the four 642 bases, changes in heli' geometry
3M-to-5M proofrea!ing: e'onuclease acti%ity of 642 polymerases
- ATP $ri+en #elicases: unwin! the template 642 heli' at replication for"s, use 2,N to
perform f'n, most of the time are boun! to inhibitor but get calle! o%er by initiator protein bin!ing
to replication origin an! lose inhibitor !uring repl#
- <ingle-stran! 642-bin!ing (<<D* proteins: pre%ent formation of hairpin loops
- PFlampP proteins "eep 642 polymerases attache! to 642 while they are mo%ing
- 642 Nrimase synthesies short (about 10 nucleoti!es* /42 primers on lagging stran! - 642
polymerase synthesies 200 (eu"aryotes* or 2000 (bacteria* nucleoti!es-long stretches of 642
(K"asa"i fragments* - /42 primers !igeste! away, empty spaces fille! in, 642 ligase seals
nic"s
- 0in!ing problem (one complete turn about the a'is of the parental !ouble heli' e%ery 10 base
5
pairs*
- sol%e! by topoisomerases: topo$ opens up one stran! of 642 by utiliing a tyrosine acti%e site
an! co%alently attaches to 642 phosphate, brea"ing phospho!iester lin"age# (.nergy is retaine!
in bon! with tyrosine*# Knce bon! is bro"en, 642 can rotate aroun! other unbro"en an! relie%e
tension# ,he stran! of 642 is then re-forme! with retaine! energy#
Replication for8s (special se=uences of 642 present at their center* 9 /eplication for"s can be
uni!irectional or bi!irectional (usually bi!irectional*
- /eplication bubbles7replication origins (up to 300 nucleoti!es long*, bin!ing of initiator proteins,
then helicase, then primase 9 primosome ; comple' forme! when initiator, helicase, an! primase
are all boun! to template 642 , then 642 polymerase can bin! an! begin synthesis base! on
primer)
- well characterie! in bacteria an! in yeasts (2/< se=uences*
- in higher eucaryotes replication origins acti%ate! in clusters (units*
- 6ifferent regions of each chromosome acti%ate! in a repro!ucible or!er !uring < phase
- <ynthesis of new histones !uring <-phase of the cell cycle8 pac"aging of new histones
- <pecial problem of the ,elomeres (L-rich repeats a!!e! to chromosome en!s by telomerase*
9 ,elomerase ; 642 polymerase with boun! /42 template: enyme that a!!s nonsense bases
to 3> en! of parental stran! (using its own /42 subunit as a template* so that 642pol can use
that as a template an! ma"e sure that replication of parental stran! continues through all
necessary bases (from 5> to 3> en! of new stran!*
- 642 re-replication bloc" (licensing factors*
TRA)SCR6PT63) , S9)T(ES6S 34 mR)A - highly selecti%e process
Str%ct%re of a :t"pical: gene : Nromoter - bin!ing of eucaryotic /42 polymerases to protein-
642 comple'es (transcription factors* - enhancer elements, upstream se=uences, ,2,2 bo'
(,2,2 factor*, silencers, position effects
- ,2,2 bo' ; se=# within promotor that eu"# /42N recognies an! transc# starts about 20 bp
!ownstream
- acti%ator protein can bin! to enhancer upstream of promotor to upreg# ,ransc# (repressor !oes
opposite*
- Lenes in pieces : 6ntrons an! E.ons#
- only about 1: of chromosomal 642 transcribe!
- start7stop signals for transcription8 stop signals (in .# coli*: self-complementary region Q run of
G
C
6ifferent types of /42 : at least three type of /42N in eu"s)
Ri!osomal R)A &rR)A' 9 ma!e in nucleolus (e'cept 5<*, foun! in ribosome>s (B0: all /42*
Transfer R)A &tR)A' 9isoaccepting, at least one per amino aci!
5essenger R)A &mR)A'
-we thin" of m/42 as being most common, but is really only 3-5: of total /42 in cell
- 5> caps (important for bin!ing to ribosomes 9 translation*, poly 2 tail (important for stability but
not present in all, absent in histones 9 why histones are only synthesie! in < phase right before
they>re re=uire!*
- introns, e'ons, ma!e into proteins by ribosomes
R)A pol"merases - bin! to promoter regions (specific 642 se=uences, oriente!, !etermine
which stran! of 642 is use! as template /42N can wor" in either !irection, but in reality only
wor"s on one stran! bc of polarity of promoter*
- bacterial - eucaryotic /42 polymerase comple' (multiple subunits, regulation*8 some bacterial
%iruses enco!e much simpler /42 polymerases
Bios"nt#esis of R)As: opening of 642 stran!s, 5M to 3M !irection of /42 synthesis (rea!s
template 642 3>-5>*
Kne /42 polymerase in bacteria - !ifferent subunits, sigma factor, elongation factors
T#ree R)A Pol"merases in e%8ar"otes: all re=uire acc# Nroteins to f'n
/42 Nolymerase $ - ma"es large ribosomal /42s - unaffecte! by alpha-amanitin
/42 Nolymerase $$ - ma"es m/42s - %ery sensiti%e to amanitin
/42 Nolymerase $$$ - ma"es t/42s, 5< /42 of ribosomes, an! other small, stable /42s 9
mo!erately affecte! by amanitin
- a!!itional initiation proteins re=uire! for bin!ing of eucaryotic /42 polymerases to promoters
(,ranscription factors* alrea!y boun! to 642
- 2ssociation of 642 with histones must be altere! (they remain in nucleosomes !uring
transcription* 642 must be opene! up somewhat, cannot use 30 nm fiber
- 6ifferent /42 Nolymerases recognie !ifferent start signals
- /42 Nolymerase $$$ - re=uires 5< transcription factor - which recognies 642 se=uences in
the mi!!le of the gene
- /42 Nolymerase $$ transcribes !ifferent se=uences at %ery !ifferent rates
- /42 synthesis %isualie! by ,.3 of sprea!s - globular /42 polymerase RFhristmas treeS
- origin of replication on 642 is locate! right before where /42 stran! is shortest (on stran!
being transc# by multiple ribo*
T
-termination signal is locate! Hust after longest /42 stran!
- !ots at en! of /42 molecules ; spliceosomes which shows that processing begins on 5> en!
before 3>en! is synthesie!
Stages of R)A s"nt#esis , nee! 2,N to start 246 en! transc#
1* interaction of /42 polymerase with 642 to form a binary comple'8
2* $nitiation of /42 chains8
3* .longation (can be !i%i!e! in at least 5 substeps*8
E* <electi%e termination - re=uires accessory proteins, 2,N#
- m/42 synthesis: heterogeneous nuclear /42 (hn/42* is imme!iately boun! to proteins
(hn/4N particles*
- all /42 e'cept t/42 !oes 4K, li"e to be na"e!, so as soon as it>s synth# $t bin!s proteins
- formation of spliceosomes
- Fappe! at 5M en!: TM3e guanosine-NNN-27L (will me!iate bin!ing to ribosomes, stabilies
m/42*
- ,aile! at 3Men!: poly-2 tail a!!e! by poly-2 polymerase (22G222 signal 20-30 nucleoti!es
upstream of clea%age: instructions to 266 tail are enco!e!, but actually poly 2 se=# not
enco!e!*8 transcription continues until termination signal reache!8 /42 fragment rapi!ly
!egra!e!: signal to a!! Noly 2 tail comes before termination signal, so /42N "eeps transc# after
poly 2 tail has been a!!e! by Noly2 polymerase, but any /42 after tail gets !egra!e!
- poly-2 tail ai!s in e'it from nucleous, gi%es stability in cytoplasm, re=uire! for efficient
translation, poly 2 bin!ing proteins bin! to tail to confer stability
-poly2 tail ne%er a!!e! to histone m/42>s because !on>t nee! to lea%e nucleus, an! nee! to be
unstable so they can be !estroye! following < phase
- primary transcript (a%erage length for m/42s : B000 nucleoti!es*# 5ery unstable - re!uce! to
a%erage length of 1200 nucleoti!es within 30 min
- m/42 processing (splicing, remo%al of introns*
- spliceosomes contains small /42s that hybri!ie specifically to se=uences to catalye /42
splicing (recognie! !onor-acceptor Hunctions*, small /42s in%ol%e! (sn/4Ns*, /427/42
pairing, lariats ; where /42 being remo%e! loops out
- 2lternati%e splicing of the same primary transcript can generate !ifferent proteins
- Fhanges in !e%elopment, !ifferent tissues: !epen!ing on time in !e%# Kr which type of tissue
the cell is, !ifferent introns get splice! an! !ifferent e'ons get put together, an! you get !ifferent
gene pro!ucts
B
- ,ransport of m/42s to cytoplasm !elaye! until splicing is complete an! highly regulate! by
nuclear pore acceptor comple'es# Nroteins associate! with /42 change after entering
cytoplasm#
Posttranscriptional control of gene e.pression
1# attenuation (/42 synthesis* ,ranscriptional control
2# m/42 processing in the nucleus
3# retention in the nucleus (e'port to cytosol*
E# m/42 localiation in the cytosol (signals in the 3M untranslate! region* is poly2 tail there1
5# translational controls
C# m/42 !egra!ation in the cytosol 9 some ha%e halflife of minutes, others stable for !ays
- can control gene e'pression after m/42 ma!e by "eeping m/42 in nucleus longer or fore%er,
not ma"ing it, "eeping it isolate! in cytosol, not translating it, !egra!ing it, e!iting it, controlling
protein pro!uct
T#e )UCLE3LUS : by ,.3 recogniable as an aggregate of closely pac"e! filaments (ribosomal
/42*, !ense granules (almost finishe! ribosomes*, amorphous matri', associate! chromatin
Site of Ri!osomal R)A s"nt#esis (not 5< /42* by Nol $ an! assem!l" of Ri!osomes
3ultiple ribosomal /42 genes, arrange! in tan!em - PFhristmas treeP appearance in sprea!
chromatin preparations: nucleolar constrictions are regions of 642 where there are hun!re!s of
copies of nuc# Krg# regions# (nee! many copies to ma"e many r/42s =uic"ly* an! Fhristmas
trees result from r/42 being synth# 2t each copy simultaneously
- 4ucleoli ma!e up of nucleolar-organier regions of chromosomes (1 or more*
- ?arger /42 gene initial transcript that is processe! to get U r/42 genes# ,he 5< /42 gets
together with the other 3 along with ribosomal proteins that are ma!e in the cytoplasm but
importe! into the nucleolus
- Nresent on fi%e chromosomes in humans (10 in !iploi! state*
- 4ucleolar- Krganier regions of !ifferent chromosomes meet in same region of the nucleus
- the parts of the chr# that are nucleolar organiing co!ing regions (2V2 642 co!ing for r/42*
are all stic"ing into same region of nucleus become the nucleolus
- no nucleolus in mitotic cells because the 642 is super con!ense! an! no transc# of r/42
ta"ing place
- S"nt#esis of tR)A;s : Flo%e-leaf structure - about 1007cell in eu"aryotes - Nrocessing of
W
t/42s pac"aging - processing of E5< r/42 precursor
- nucleolin: an abun!ant /42-bin!ing protein apparently only associate! with r/42s
- K%erall scheme of ribosomes synthesis
- 5< r/42 is co!e! for an! transc# outsi!e of nucleolus (on chromosome 1*, so must be
mo%e! into nucleolus to be a!!e! to immature subunits as it>s assemble!
- <mall an! large immature subunits lea%e nucleolus an! become mature subunits in nucleus
- mature small an! large subunits lea%e nucleus separately an! assemble when 4..6.6 in
cytosol (when m/42 bin!s small subunit*
)UCLEAR E)7EL3PE : 2 membranes about 20-E0 nm apart, composition an! function !iffer:
- $nner membrane organies chromosomes, bin!s to fibrous lamina proteins8
- Kuter membrane, similar an! contiguous to ./, ribosomes are often present
- Nerinuclear space in between, contiguous with lumen of /./
)UCLEAR P3RES (nuclear pore comple'es* : sites of e'changes between nucleus an!
cytoplasm, about W nm a=ueous channels
-pores are highly selecti%e but small molecules (ions, 2,N, Ni, etc* pass freely through
- test this using non-nuc# proteins (bc "now nuc# has no mech# to transport them alrea!y*
-for prot# 1-2 nm, go freely8 2-3 nm, ta"es longer8 3-5 nm, ne%er
Sc#eme of n%clear pore :
- 2 rings of B subunits each, one on cytoplasmic si!e an! other on nuclear#
- 2 rings connecte! by B column subunits (bul" of the pore wall*
- $n the center ha%e B annular components (e'ten! spo"es towar! center* where receptors are
- 2t cytoplasmic si!e ha%e fibrils e'ten!e!
- Kn nuclear si!e ha%e nuclear cage e'ten!ing into the nucleoplasm pre%ents chromatin from
clustering in the area an! clogging pore
- 6istribution of nuclear pores usually homogeneous, but in some cases clustering
!emonstrate!, reflecting cell structure an!7or acti%ity 9 usually clustering occurs when the cell is
%ery acti%e in transport of m/42 an! ribosomal subunits
A))ULATE LA5ELLAE
- fragments of nuclear en%elope an! nuclear pores floating in cytoplasm, li"ely bc there were too
many pores in the membrane an! nee!e! to get ri! of some1
10
- in the cytoplasm of some germ cells (fre=uent in oocytes* 9 not common among most cell types
- foun! in some tumor cells
- function un"nown, but !emonstrate that nuclear pores can e'ist elsewhere than on nuclear
en%elope
- these pores get !isassemble! by cell !uring mitosis Hust li"e the pores in the nuclear en%elope
(because en%elope gets !egra!e!* same mechanism affects all the pores
- 3o!el of 2nnulate ?amella
BioAP 3160 - LECTURE 6
E<C(A)=E 34 5ATER6AL between nucleus an! cytoplasm: only small molecules (ions, etc#*
an! small protein (up to about E5,000 !altons* can mo%e freely across nuclear pores8 passage
highly regulate! for larger molecules (ribosomes, for e'ample* which cannot freely an! passi%ely
!iffuse in7out of nucleus (pores too small* - acti%e process re=uire! - specific receptors
Proteins acti+el" transporte$ t#ro%g# n%clear pores: nuclear import signals on specific
proteins (short pepti!es, E-B 22 long*8 e'amples: <5E0 ,-antigen (%iral protein that has to get in
nucleus to be function, has specific se=uence of 522 ?ys-?ys-?ys-2rg-?ys 9 if change! protein
can>t get into nucleus*, nucleoplasmin
- highly specific short 22 signals8 w7 proper signal, see almost no nuclear proteins in cytosol
- protein transport through the pores can be regulate! (e#g# glucocorticoi! receptor7hspW0*
- "aryophorins ; nuclear transporter (many "in!s*, wor" closely with small, monomeric L proteins
- /an subclass of L,N proteins is in%ol%e! in nuclear transport:
/an-L2N (L,Nase acti%ating protein, L,N L6N* is only in cytosol so cytosol has
mainly /an-L6N#
/an-L.F (Luanine e'change factor, L6N L,N* is only in nucleus so nucleus has
mainly /an-L,N
Lra!ient !ri%es nuclear transport
$mport
o $mportin bin!s cargo in cytoplasm an! pases through nuclear pore
o 0hen reaches nucleus, /an-L,N bin!s an! importin releases cargo
o Decause /an-L6N in cytosol !oesn>t bin! cargo receptor, unloa!ing only happens
in nucleus 9 !irectionality
o .mpty receptor with /an-L,N is transporte! bac" to cytosol where it is hy!rolye!
bac" to /an-L6N so it can begin ne't cycle
.'port
o /an-L,N in nucleus promotes cargo bin!ing to e'port receptor rather than
!issociation
o Knce e'port mo%es through pore to cytosol, its /an-L,N is hy!rolye! to /an-L6N
causing release of cargo an! /an-L6N in cytosol#
o .'port receptor is returne! to the nucleus to complete cycle
- selecti%e transport of /42s through nuclear pores: a role for the 5Mcap of m/42s
11
- area of rarefaction often present at nuclear si!e in correspon!ence with nuclear pores
46BR3US LA56)A (4uclear ?amina* 9 ?amins are attache! to inner en%elope membrane, most
conspicuous in some cells of in%ertebrates, but present in almost all cells that ha%e a nucleus
- in mammals compose! of three uni=ue proteins, C0-T0 "6a, calle! lamins
- lamins ma"e up one class of interme!iate filament proteins
-all E classes (lamins, "eratins, neurofilaments, an! !esmin7%imentin7glial fibrillary ha%e common
alpha helical region, but lamins ha%e small D sheet "in" in that region
- !ifferently from other filament proteins, lamins contain nuclear transport signal (to get into
nucleus*
- assemble in a two-!imensional lattice (other proteins in%ol%e! 1*, %ery !ynamic
- lamins regulate !issolution of nuclear en%elope in preparation for mitosis, cycle of
phosphorylation7!ephosphorylation - formation of tetramers of lamins 2,F Q membrane-boun!
lamin D - !istribute! in cytoplasm !uring mitosis - !ephosphorylation at beginning of interphase
with re-formation of nuclear en%elope
-phosphorylation of lamins by F6V>s causes !issolution of nuclear en%elope
-!ephos# at beginning of interphase in new cell causes reformation of en%elope
- fibrous lamina also thought to interact with chromatin, an! to gi%e shape - stability to the
nuclear en%elope
- possibly helps control which chromatin e'press when1 un"nown
R6B3S35ES A)D PR3TE6) S9)T(ES6S
Protein S"nt#esis : Lenetic co!e: 20 aminoaci!s, C1 co!ons (3 are stop co!ons : G22, G2L,
GL2*
- 6etermination of rea!ing frame: initiation co!on (2GL ; methionine*, Voa" consensus
se=uence
- 22>s are a!!e! to the carbo'yl-terminal en! of a growing polypepti!e chain
- ,he e%ents in protein synthesis are catalye! on the ribosome
T#e Translational 5ac#iner" : components an! mechanisms#
- m/42 (nee! 5> an! 3> nonco!ing se=# for transport out of nuc# an! rec# by ribo*
- aminoacyl 9t/42s (acti%ation of 22, coupling with m/42*
- ribosome (small, large subunits, 2, ., N sites*
- soluble protein factors (initiation, elongation, termination*
Aminoac"l-tR)A;s - Aminoac"l-tR)A S"nt#etases
- 2minoacyl-t/42Ms perform two functions :
1* they acti%ate the amino aci! for pepti!e bon! formation8 antico!on somewhere in mi!!le
2* the t/42 portion acts as an Pa!aptorP between the m/42 an! the amino aci! on the ribosome#
- Specificit" of the machinery :
12
a* t/42s 8 ha%e at least one t/42 for each 22, must a!! correct 22 to t/42 with proper anti-
co!on
b* aminoacyl-t/42 synthetases: ha%e at least one aa-t/42 synth for each aa-t/42 couple,
bon! between 3> en! of t/42 an! 22 is high energy, its clea%age pro%i!es energy for pepti!e
bon! formation
S"nt#esis of aminoac"l-tR)As : in two steps (specific enymes )*
22 Q 2,N ; a!enylate! amino aci! attach 2,N to 22
2!enylate! 22 Q t/42 ; aminoacyl-t/42 Q 23N use 2,N to attach aa22 to t/42
Ri!osomes : similar in pro"aryotes (T0<* an! eu"aryotes (B0<*
- $n mitochon!ria an! chloroplasts (an! in pro"aryotic cells* present as T0< ribosomes,
!issociating into 30< Q 50< subunits#
- B0< ribosomes sensiti%e to cyclohe'ami!e, T0< ribosomes to chloramphenicol#
- <mall Q large subunits assemble! only !uring the initiation process after an m/42 has boun!
to small subunit
- 6istinct groo%es for nascent polypepti!e (30 22s* an! for m/42 (35 nucleoti!es*8 P Site
(pepti!yl-t/42 bin!ing site* an! A site (aminoacyl-t/42 bin!ing site*
Nrotein factors : 3ost of them act while boun! to the ribosome: initiation, elongation, an!
termination factors#
<teps in protein synthesis:
6nitiation (.u"aryotes* :
1# 6issociation of B0< ribosomes into subunits8
2# 2cti%ate! 3et-t/42i (initiator t/42* bin!s to E0s subunit, boun! to N-site on small subunit8
e$F-2 re=uire! for proper positioning (rate controlling factor in some cells*8 forme! stable
ternary comple' with e$F-2 an! L,N (which is not hy!rolye!*
3# m/42 bin!s to E0< subunit - promote! by %arious factors - 2,N is hy!rolye! - /esult:
>0S pre-initiation comple.# 5M cap on m/42 !irects bin!ing of small ribosomal subunit to
5M en! of m/42 - subunit then mo%es along m/42 in search of first 2GL start co!on
E# C0< ribosomal subunit bin!s to E0< pre-initiation comple'8 L,N hy!rolye!, factors
pre%iously boun! are release!#
Elongation - !i%i!e! into 3 phases :
1# bin!ing of aminoacyl-t/42 to P2P site
2# pepti!e bon! formation
3# translocation- ribo an! m/42 mo%e with respect to each other
/eactions occur on the surface of the ribosome an! in%ol%e 2 an! N sites catalye! by Nepti!yl
transferase (a structural part of the large ribosomal subunit*
13
Termination - Nrocess begins with pepti!yl-t/42 in the N site, an! one of the three nonsense
co!ons (G22,G2L, GL2* in the 2 site# ,his configuration is recognie! by appropriate release
factor (/F* which bin!s to the ribosome (process re=uires L,N in eu"aryotes*# Nresence of /F
acti%ates pepti!yltransferase center, which transfers pepti!yl moiety to water FKKH# ,he
%arious macromolecules then !issociate from the ribosome#
Pol"somes : se%eral ribosomes boun! to the same m/42 molecule - common feature
Proofrea$ing processes
- recognition of incorrectly loa!e! aminoaci!s by aminoacyl-t/42 synthetases (2 acti%e sites on
each enyme* incorrectly paire! t/42>s preferentially !issociate
- .F-,u boun! to t/42, after the initial co!on recognition, hy!rolyes its boun! L,N, before
!issociating from the ribosome lea%ing the t/42 in place
-Vinetic !elay: if it>s the wrong t/42, the hy!rolysis of L,N ta"es long enough that ribosome has
time to recognie wrong t/42 an! !issociate it
- 6elay between co!on-antico!on pairing an! polypepti!e chain elongation allows time for
!issociation of incorrect t/42Ms
Reg%lation of protein s"nt#esis
- $mportant to !etermine proteins synthesie! by specific cells at !ifferent stages of !e%elopment7
!ifferentiation, in response to hormones an! other factors, etc#
- <e%eral mechanisms an! factors in%ol%e! !escribe!, but !etails often still unclear#
- Nhosphorylation of protein factors (e$F-2*, an! of E0< subunit (by c23N-!epen!ent protein
"inase*8
- polyamines: affect rate an! selecti%ity of m/42
- Fontrol in reticulocytes by heme eif-2 phosphorylation
- 5irus-in!uce! shut-off of synthesis of host proteins
o 5irus-in!uce! mo!ification of initiation factors or altere! re=uirement for the factors8
o 5irus-in!uce! changes in intracellular ionic composition
o /ibosome mo!ifications - 5irus ma"es ribosome>s select only %iral m/42 an! not
intrageneous m/42 host genes are not e'presse!
5an" %sef%l anti!iotics are in#i!itors of procar"otic protein s"nt#esis
-some antib>s that wor" on eu" an! pro" cells can be use! in humans $F human cell membranes
are impermeable to the !rugs
BioAP 3160 - LECTURES ?-11
T(E C35PART5E)TAL6@AT63) 34 CELLS - T#e c"tosolic compartment
- 3aHor intracellular compartments in a typical cell !efine! by !ifferent membrane-boun!e!
organelles
- ,he cytosol - about half of the total cell %olume - organie! by filaments (cytos"eleton*
- /api! !estruction of selecte! cytosolic proteins %ia the ubi=uitin-!epen!ent pathway
1E
Fytoplasmic proteins ha%e highly %ariable lifetimes 9 why1 GD$XG$,$4
Nroteins that nee! to be !egra!e! by proteosomes in the cytosol are tagge! with many
Gbi molecules
Nrocess in%ol%es ubi=uitin-acti%ating enyme that bin!s ubi=uitin to ubi=uitin ligase
<ome proteins ha%e !egra!ation signal - se=uences that e'press they>re rea!y to be
!egra!e!, usually in 4 terminal#
,he Gbi-ligase comple' recognies this signal an! transfers ubi=uitin to the protein to be
!egra!e!# 3ultiple ubi=uitins a!!e! chain
Nroteosome is attracte! an! !egra!es protein
Heat-shoc" an! stress-response proteins an! their role in protein fol!ing (molecular chaperones*
Fhaperones bin! unfol!e! or attempting to fol! proteins an! pre%ent them from fol!ing
improperly or prematurely, an! then help them to fol! when time is appropriate
- 4 terminal region can fol! before carbo'yl has finishe! being synthesie!, but to ha%e
proper fol!ing nee! synthesis of both en!s first so can ha%e 4-F interactions
- Fhaperones associate with proteins being synthesie! an! "eeps them unfol!e! to gi%e
them time to be complete! before it fol!s
- /e=uires 2,N to assoc# an! !issoc#
- HspT0 most common chaperone in cytosol 9 calle! heat shoc" because !efense
mechanism to remo%e improperly fol!e! proteins that ha%e been synthesie! because of
poisoning, etc#
- Hsp C0 an! 10 in mitochon!ria
- DiN in ./ lumen
STRUCTURE A)D 4U)CT63) 34 B63L3=6CAL 5E5BRA)ES
- <electi%e permeability barriers 9 allows cell to maintain !ifference in inner7outer en%ironment
- <ite of selecti%e transport systems
- <upport for catalytically acti%e molecules, an! for protein synthesis (intracellular* (./*
- Nart of energy trans!ucing systems (mitochon!ria*
- Fon!uction of electrical impulses (in ner%e an! muscle*8
- $nsulator function (myelin sheath*8
- <ite of specific a!hesion an! attachment cell components8
- <ite of cellular components in%ol%e! in cell recognition8
15
- <ite of hormone receptors, an! for the transfer of their signals insi!e the cell8
- $ntercellular communication8
- 2lso, ./ an! mito membranes ha%e T times the <2 of plasma membrane
- $n highly acti%e cell, more ./ membrane will be present
5et#o$s %se$ to st%$" mem!rane str%ct%re
1# Gltrastructural an! morphological e'amination : ,.3, <.3, freee-fracture replica techni=ue
2# <ubcellular fractionation an! isolation of specific membrane fractions
3# Diophysical techni=ues : !etermination of P,ransition temperatureP, .lectron <pin /esonance,
4uclear 3agnetic /esonance, etc#
E# Diochemical analysis of membranes an! membrane components : analysis of lipi!s an!
proteins, phospholipase !igestion, etc#
5E5BRA)E C35P3)E)TS , L6P6DS
- Nhosphati!yl -choline, -inositol, -ethanolamine, -serine
- 6eri%ati%es of sphingosine : sphingolipi!s, glycolipi!s
- Fholesterol : regulates flui!ity, confers mechanical stability
All A5P(6PAT(6C : ha%e polar hea! groups, hy!rophobic tails
- $mportant features of phospholipi!s : !ifferent lengths of fatty aci!s an! presence of unsaturate!
cis-6ouble bon!s ("in"s - !ifferences in flui!ity*
- Nhospholipi!s spontaneously form bilayers - hi!e nonpolar regions from water - High mobility on
plane of bilayer but flip-flop %ery rare8 Fan be reconstitute! into artificial %esicles (liposomes*
- 5ariability in lipi! composition of membranes
- Llycolipi!s carry sugar chains with sometimes %ery comple' structures (bloo! groups, L31-
receptor for cholera to'in, other cell-cell communication functions1 receptors for hormones 1*
-?ipi! rafts: clumps of lipi!s within RseaS of intermembrane space, contain high conc# of acylate!
an! LN$ anchore! proteins, present in apical polarie! membrane of epithelial cells
5E5BRA)E C35P3)E)TS , PR3TE6)S
- 5ariability in protein content of membranes - ?ess than 25: in myelin, up to T5: in some highly
specialie! membranes, about 50: of the mass in most membranes 9 /DF>s are the only type
with spectrin cytos"eleton, most cells ha%e myelin#
- ?ipi! bilayer ser%es as a sol%ent for membrane proteins - specific interactions between certain
lipi!s an! membrane proteins
1C
- Neripheral %s integral membrane proteins - 2mphipathic nature of integral membrane proteins
!ue to the presence of regions interacting with lipi!s (either spanning the bilayer or immerse!
only in half of it - membrane anchors*
C%rrent mo$el of mem!rane str%ct%re , t#e 4LU6D-53SA6C 53DEL ,
- .mphasis of this mo!el is on the great !egree of flui!ity of the proteins in membranes# $ntegral
membrane proteins embe!!e! (spanning* the phospholipi! bilayer, an! relati%ely free to mo%e in
the plane of the bilayer
- ,hings can mo%e in plane of bilayer, but are sometimes anchore! to cytos"eleton)
- Flipping bac" an! forth is highly regulate! by flippases an! scramblases
- Neripheral proteins not !irectly associate! with the membrane, but with integral proteins
- AS955ETR9 34 5E5BRA)ES : sugar resi!ues only on outsi!e (surface membrane* or
insi!e intracellular membranes8 also !emonstrate! asymmetry in lipi!s composition - $nteractions
of membrane proteins with the cytos"eleton
- once inserte!, lipi!s an! proteins ha%e polarities within their leaflet or across bilayer
E)D35E5BRA)E &E5' S9STE5 A comm%nication !et*een mem!rane s"stems
-2lso calle! the <ecretory Nathway
-3embers of the .3 system are the cell membrane, secretory %esicles, lysosomes, early an! late
en!osomes, the Lolgi, an! the ./
- ./ has proteins inserte! co-translationally
-Ha%e forwar! (sec* an! re%erse (recycling* pathways which are both always functioning
-4uclear membrane, mitochon!ria, pero'isome, an! plasti!s: 4K, members of .3 system 246
all ha%e their proteins inserte! post-translationally
-$n %esicular transport, membrane components of !onor compartment become components of
target compartment
T(E E)D3PLAS56C RET6CULU5
3any %ital biochemical processes ta"e place in or on membrane surfaces# $nternal membranes
!i%i!e the cell into specialie! compartments# $n most cells, particularly those acti%e in protein
secretion (secretory cells* because secretory proteins are ma!e on the /./, the .n!oplasmic
/eticulum represents a maHor portion of the total cell membranes#
- ./ stu!ie! by both ,.3 an! subcellular fractionation techni=ues# /esults obtaine! sometimes
1T
conflicting
- For reasons which will become ob%ious later, the ./ has been !i%i!e! into /ough (/./* an!
<mooth (<./* .n!oplasmic /eticulum : their membranes are almost i!entical in composition,
but ha%e =uite !istinct functions
RER
- 6escribe! originally as a set of filamentous structures, which staine! intensely with basic !yes
(basophilic* by light microscopy# Falle! .rgastoplasm (term now rarely use!*
- ,.3 obser%ations !emonstrate! the presence in cytoplasm of membrane-limite! cisternae,
stu!!e! with ribosomes only at their cytoplasmic surface# ,he ribosomes ma"e the /./
basophilic (/42* an! represent the main !ifference between /./ an! <./
- /./ %ery abun!ant in secretory cells (secretory proteins are synthesie! on /./* - both
en!ocrine an! e'ocrine cells usually ha%e large amounts of /./
- .stimates of the total surface area occupie! by the membranes of the /./ is up to B,000
um7cell (guinea pig pancreatic acinar cells*, representing 273 of the total area of membranes in
the cell
- 36 configuration of /./ : fenestrate! cisternae, anastomosing tubuli, isolate! %esicles8
cisternae may be arrange! in parallel stac"s 2-E um long, or may form e'tensi%e concentric
systems8 they enclose the lumen of the /./
- 2symmetric !istribution of ./ enymes - important ) membrane proteins (an! also lipi!s* are
%ery asymmetric gi%es ./ its !istinct f'n
- /./ cisternae continuous with outer nuclear membrane
-/./ lumen continuous with intermembrane space of nuclear en%elope
- Nurification of subcellular fractions enriche! in ./ components - !ifferential centrifugation 9
useful for separating /./ an! <./ an! ribosome>s from /./ because of their !ifferent !ensities
- Fractions rich in 3 ribosomes originally calle! P3icrosomesP8 correlate! biochemical an!
ultrastructural stu!ies establishe! the i!entity of microsomes an! en!oplasmic reticulum
(microsomes are obtaine! as an artifact of subcellular fractionation by fragmentation of the ./
!uring Homogeniation an! resealing of the fragments into %esicles*
-/./ ma"es all the secretory proteins of the cell an! all the .3 system members> membrane
proteins
-2lso the site of the first step of glycosylation of glycoproteins
SER
1B
- <et of membrane-limite! tubules - continuous with /./ but !e%oi! of ribosome
-,ubules loo"s a lot li"e %esicles in cross section, har! to !istinguish the two
- 0ell !e%elope! in some cells, but !ifficult to i!entify by ,.3 in many cell types
- Diochemical properties of /./ an! <./, apart from the !isparity in ribosomes content, %ery
similar# <./ an! /./ ha%e all the same enymes, localie! similarly
- Highly %ersatile organelle %ery well !e%elope! in cells in%ol%e! in acti%e metabolism of lipi!s or
metabolism of 'enobiotics (in!uction by phenobarbital in li%er*
- <teroi! secreting en!ocrine glan!s: often present as e'tensi%e spiral or concentric arrays of
fenestrate! cisternae organie! aroun! lipi! !roplets 9 <./ pro!uces steroi!s
5ain f%nctions of RER
- <ynthesis of membrane proteins an! secretory proteins (translocate! into the lumen of the
/./ as they are synthesie! by membrane boun! ribosomes*
- glycoprotein synthesis 9 first step of glycosylation of glycoproteins
5ain f%nctions of SER
- <ynthesis of lipi!s (cholesterol, phospholipi!s, triglyceri!es, steroi!s* 9 <./ enlarge! in steroi!
pro!ucing cells# 2ll membrane lipi!s of cell originate in <./
- !egra!ation of glycogen
- !eto'ification processes (!rugs, steroi! hormones, carcinogens, etc* 9 <./ mo!ifies lipophyllic
wastes that are har! to enter bloo!stream so they are water soluble#
- transport of ingeste! lipi!s insi!e intestinal cells
-FaQQ se=uestering an! release (important in signal trans!uction pathways*
-Nhospholipi! synthesis always begins in the cytoplasmic leaflet of the ./ membrane
- <tarts with fatty acyl Fo2 an! glycerol 3N (acyl transferase* phosphati!ic aci!
(phosphatase* !iacylglyercol (choline phosphotransferase* phosphati!yl choline
phospholipi! a!!e! to cytosol half of lipi! bilayer
- $f outer leaflet is always growing, how !o we get the two to e%en out1 ,/24<?KF2,K/<
-,ranslocators e'plain the symmetry of the o%erall 4G3D./ of lipi!s in each layer an! the
asymmetry of the ,@N.< of lipi!s in each layer
T(E S6=)AL (9P3T(ES6S &%ni+ersall" accepte$'
-.'plains the association of specific ribosomes an! their attache! m/42>s with ./ membrane 9
1W
cotranslational import into lumen of ./
1# <ecretory an! membrane proteins are ma!e by ribosome>s boun! to ./ 9 ribosomes only
attach when synthesiing
2# Free an! boun! ribosome>s can e'change subunits 9 no !ifference
3# Free an! boun! ribosome>s are i!entical 9 what is !ifferent is pro!uct of translation ; signal
E# Nro!ucts of protein synthesis in /./ membrane are always segregate! in a compartment,
ne%er in the cytosol (./, Lolgi cisternae, Lolgi %esicles, out of cell*
5# 6uring or shortly after translation in /./ membrane, certain mo!ifications can be ma!e in the
./ lumen#
C# <ignal hypothesis: information !etermining the association of certain ribosome>s with the /./
membrane is containe! in the 4-terminal segment of the protein 9 t#e Bsignal pepti$eC
- ,his is all true, but not totally complete because now "now that signal is sometimes internal an!
not 4-terminal
(3D S6=)AL PEPT6DES ARE UT6L6@ED
-<ecretory protein synthesis always begins at a free ribosome in the cytosol
-2bout 25-30 22 into synthesis, the signal pepti!e is re%eale! (pre%iously hi!!en by large
subunit*
- ,he <N is recognie! by the signal recognition particle (</N* which is ma!e of protein an! /42
- Din!ing of the </N temporarily halts translation an! gi%es the free ribosome time to mo%e to the
./ membrane an! fin! an </N receptor an! a translocator
- ,he </N receptor recognies the <N-</N-ribosome comple' an! bin!s the ribosome
- ,he </N receptor bin!ing the ribosome helps to gui!e the pepti!e to a channel in ./
-once the polypepti!e lin"s the ribosome to the ./ , the </N-</N receptor !issociates
- translation continues until pepti!e fully synthesie! an! signal pepti!ase clea%es <N, which then
remains in ./ membrane
-<N either gets !egra!e! in ./ membrane K/ if the <N is not completely, can become
membrane boun! part of ./ membrane protein
ER 5E5BRA)E PR3TE6)S, S6)=LE A)D D3UBLE PASS
-<ingle pass (class 1* proteins ma!e by:
start transfer se=uence (<N* an! stop transfer se=uence (stops protein from entering
lumen of ./*
translation continues until stop transfer se=uence is reache!
20
F-term of pepti!e stic"s out into cytosol
<tart transfer se=uence is clea%e! an! 4-term of pepti!e stic"s into ./ lumen
Fan become anchore! in LN$-anchor of lipi! raft
-6ouble pass proteins ma!e by: multiple start an! stop transfer se=uences# <ame as single
e'cept start transfer se=uence ne%er clea%e! an! now protein has 2 transmembrane segments
ALL PR3TE6) S9)T(ES6S STARTS 3) 4REE R6B3S35ESEE
PR3TE6) ASSE5BL9 F DE=RADAT63) 6) T(E ER
- Translocate$ pol"pepti$e c#ains fol$ in t#e l%men of t#e ER
- 2s pepti!e is synthesie!, it is boun! by chaperones in the ./ lumen to pre%ent fol!ing before
the entire thing is synth# then they help it fol! once it>s !one synth#
- Nrotein !isulfi!e isomerase an! its role in correct !isulfi!e bri!ges formation
-N6$ is K4?@ foun! in the lumen of the ./ all !isulfi!e bri!ges are forme! in lumen of ./
- ma"es sense because usually only secrete! proteins ha%e !isulfi!e bri!ges an! all secrete!
proteins are ma!e in ./)
- Nepti!yl prolyl cis-trans isomerases: abun!ant an! wi!ely !istribute! - inhibite! by
immunosuppressi%e agents
-Nct$ catalyes switching between cis an! trans pepti!e bon!s in proline resi!ues
- /ole of bin!ing proteins-chaperones (DiN, stress-response proteins*
-DiN role: bin!s proteins as they enter the lumen an! pre%ents improper fol!ing, hy!rolyes 2,N
to pro%i!e energy nee!e! to pull protein across ./ membrane
- 2lso ma"es sure $g ha%e hea%y 246 light chains before they can be sent out of ./
-Falne'in an! calreticulin: chaperone proteins that re=uire 1glucose on protein to be able to bin!
- the longer a protein is in the ./, the more glucoses it loses
- if it>s still unfol!e! 246 loses all glucoses, it must be !egra!e! bc will ne%er fol! right
- if it>s still unfol!e! 246 glycosyl transferase "eeps a!!ing a glucose bac" on, calne'in an!
calreticulin will "eep bin!ing it an! helping it fol!
- /ole of 2,N bin!ing an! hy!rolysis by stress-T0 proteins: pro%i!es energy nee!e! to pull
pepti!e across membrane
- <electi%e protein !egra!ation: if the protein still isn>t fol!e! right, it is recognie! by re%erse
translocators (<ecC1 comple'* that bring it out into cytoplasm where it can recei%e a multi-Gbi tag
an! be !egra!e! by proteosome
- Llycosylation of proteins in ./: 4-lin"e! oligosacchari!es a!!e! in lumen of ./ to 2sn (4*
21
resi!ue on protein being translocate!
- ,he 4-lin"e! oligosacchari!e is built sugar by sugar in association with !olichol (lipi!* an! then
whole thing a!!e! (en bloc* to protein
TRA)S4ER of PR3TE6)S an$ L6P6DS from RER to cis-=3L=6
- ta"es place %ia %esicles, bu!!ing from the transitional elements of the /./: these are areas of
/./ where there are no ribosome an! there is a lot of blobs7proHections that will become %esicles
- $nterme$iate compartment, 2lso calle! the sal%age compartment or cis-Lolgi-networ"
- forme! by fusion of %esicles that ha%e Hust left ./
- between /./ an! Lolgi, re-cycling of resi!ent /./ proteins: if something is sent out of
transitional networ" by mista"e, the sal%age compartment recognies this an! sen!s a %esicle
bac" to the ./ with the molecule in it (usually resi!ent ./ proteins*
- me!iate! by specific receptors: these receptors are constantly cycling from ./-intm# comp#-./
e%en if they !on>t RcatchS anything in the intm# comp#
- role of V6.? an! VAAA se=uences: DiN has a V6.? se=uence, other resi!ent proteins ha%e
VAAA se=uence that bin! !irectly to FKN $ coats to be sent bac" to the ./
- ./, intm# comp# an! Lolgi !istinguishe! by particular enymes present in them
T#e =3L=6 C35PLE<
- Kbser%e! by light microscopy (Lolgi* after staining with hea%y metals
- 6iagram of Lolgi apparatus: thin in central portion, e'pan!e! at periphery8 Fon%e' - forming
(Cis* face 7 Fonca%e - maturing (trans* face NK?2/$,@
- (istoc#emical staining of t#e =olgi comple.: only seen after lea%ing prep for too long
- Trans-t%!%lar net*or8 (%isualie! in li%er cells after ?owycryl low temperature embe!!ing*
E+i$ence for $istinct s%!compartments (monensin bloc"s cis-to-trans transfers*
-Cis =olgi net*or8, interme!iate compartment Q Fis Lolgi, sorting center
-Cis-=olgi : impregnate! with osmium, phosphorylation oligosacchari!es on lysomomal proteins,
remo%al of mannose
-5e$ial =olgi : remo%al of mannose (an! glucose 1* from glycoproteins8 a!!ition of Llc42c
- Trans =olgi : staining for thiamine pyrophosphatase, staining for aci! phosphatase, a!!ition of
terminal sugars to glycoproteins, proteolysis of preproteins, sulfation of glycoproteins, a!!ition of
Lal
- Trans-=olgi-)et*or8, some in+estigators consi$er it a separate organelle
22
-,rue shipping an! pac"aging center
-3uch larger than rest of Lolgi apparatus
,ransfer of proteins an! lipi!s between Lolgi cisternae: %ia %esicles, non-clathrin-coate!, bu!!ing
from the rim of one cisterna an! fusing with the ne't (see ne't ?ecture*8 tubular connections may
be in%ol%e! in re%erse transfer (transcis*
5aGor f%nctions of =olgi comple.
- <ynthesis of glycoproteins an! glycolipi!s
- Nroteolytic processing of preproteins (,rans Lolgi*
- Nac"aging of lipoproteins an! formation of secretory granules (,rans Lolgi networ"*
- /ecycling of surface membrane components (Fis Lolgi networ" (intm# comp#**
- 3embrane biogenesis
- Fontrol center for !istribution of membrane components into !ifferent cellular membrane
fractions (,rans golgie*
=L9C3PR3TE6) S9)T(ES6S
4-lin"e! %s K-lin"e! oligosacchari!e chains
- )-lin8e$ oligosacc#ari$es: initial step $%ring translation in the /./ membrane (en bloc*
./ ?umen ; initial Lolgi
Lolgi ?umen
o Lolgi mannosi!ase remo%es three more mannoses forms high-mannose
oligosaachari!e# $mportant for mar"ing lysosomal enymes# $f not use! for lysosme,
two more mannoses remo%e! by me!ial golgi, then others a!!e!
,hree glucoses remo%e! in ./# 0hy a!! three glucoses Hust to remo%e them before it gets
out of the golgi1 Decause calne'in chaperone nee!s a glucose to chaperone#
- e'tensi%e processing later in the /./ an! in the !istinct portions of the Lolgi comple': 4-lin"e!
oligosacchari!e can become comple' (original oligo# is trimme! an! more sugars are a!!e!* or
can become high mannose (some original oligo# trimme! but no new sugars a!!e!*
- <pecific glycosi!ases clea%e !ifferent biosynthetic interme!iates
- 3-lin8e$ oligosacc#ari$es: sugars a!!e! se=uentially (no initial bloc" transfer*
Concentration of secretor" pro$%ctsH
- Foncentration is not !epen!ent on continuous e'pen!iture of energy#
23
- Foncentration (which continues !uring maturation of secretory granules* apparently results from
the formation of osmotically inacti%e aggregates: as %esicles mo%e through Lolgi networ", in the
granule positi%e an! negati%ely charge! proteins get together an! neutralie the charges# 0hen
charges are lost, there>s a change in osmotic acti%ity in granule water mo%es to cytoplasm by
osmosis# ?ose some of their cytoplasmic content (sent bac" to Lolgi in clathrin coate! %esicles*
an! the molecules within them form aggregates, water lea%es %esicle contents become
concentrate!# 4o net mo%ement of water through channels#
- Constit%ti+e secretor" pat#*a" 9 !on>t concentrate, %esicles containing (collagen,
immunoglobulins, etc* are imme!iately !ischarge! to the surface, not retaine! in secretory
granules# 6on>t nee! concentration
Secretor" =ran%le formation
Knce the secretory proteins reach the trans face of the Lolgi comple', they are further
concentrate! an! pac"age! in membrane-limite! %esicles, %acuoles, or granules#
Temporar" storage of secretor" pro$%cts in mem!rane-!o%n$ +ac%oles an$ +esicles
Gn!er normal, resting con!itions, secretory granules appear incapable of recogniing an! fusing
with each other, although in some cells they are closely oppose! to each other# ,he reasons for
this lac" of fusion are un"nown at present# (picture of resting mast cell with lots of %esicles sitting
near cell membrane*
-in or!er for contents of %esicle to be secrete!, %esicle membrane must !oc" on cell membrane
an! fuse with it to release contents outsi!e cell#
7ES6CULAR TRA)SP3RT
-,he Fonstituti%e secretory pathway: once a %esicle is ma!e an! sent to membrane it gets
secrete! imme!iately
-,he /egulate! secretory pathway: only some cells, %esicle is not release! from trans-Lolgi until
cell recei%es signal to !o so
L9S3S35ES
- 6isco%ere! by chance: irregularities in enyme assays
- 3embranous bag of hy!rolytic enymes use! for controlle! intracellular !igestion of
macromolecules to micromolecules to be reuse! in the cell
2bout E0 enymes "nown to be present in lysosomes - all are aci! hy!rolases (pH optimum
2E
about 5* - e'tra protection against acti%ity if release! in the cytoplasm (other protection ;
membrane* has 64ases, phosphatases, lipases 9 can !igest almost e%erything# .nymes
synthesie! in ./ 9 high mannose has phosphate group a!!e! in cis golgi 9 sorting mar"er so
transporte! to lysosome by 3CN receptor# 3annose CN bloc"s further processing of carb chains#
- 3embrane of lysosomes contains transport protein which utilies energy of 2,N hy!rolysis to
pump HQ into the lumen of lysosomes (HQ pump maintains low pH in lysosome*
?ysosomal membrane resistant to the enymes it encloses, an! impermeable to their substrates -
Nrocess of lysosomal !igestion carrie! out insi!e the lysosomes
- Histological staining for lysosomes
- Nrimary lysosomes often foun! clustere! aroun! the Lolgi comple'
-?ysosomal enymes wor" at pH lower than cytosolic, so that>s another !efense mechanism
against lyso# enymes getting loose an! !estroying cell
5ain role of l"sosomes : cell !igesti%e system - !igestion of intra- an! e'tracellular (lysosome
secrete! to !igest stuff outsi!e cell* !ebris, phagocytose! organisms, cell nutrition (cholesterol
assimilation*
1* 6igestion of materials ta"en up by en$oc"tosis (coate! pits, en!osomes*
2* Drea"!own of intracellular material 9 a%top#ag" 9 !uring !e%elopment an! healing
3* 6igestion of p#agoc"tose$ particles an! microorganisms - only in specialiIe$ cells
E* acrosome of sperm cell contains enymes to brea" !own oocyte cell membrane, bone
reabsorption
En$ res%lts : small 30 components enter cytoplasm an! are re-utilie!
- lipof%scin pigments: accumulate with age, e%i!ence of things not !egra!e! o%er time
- ?ysosomes in%ol%e! in many human !iseases an! syn!romes: storage !iseases
- Storage $iseases : accumulation within the cells of %arious substances (glycogen, glycolipi!s,
etc#* !ue to lac" of specific lysosomal enyme - genetic !efects 9 too much of something "ept in
cell that shoul! ha%e been !egra!e! in lysosome
S"nt#esis of l"sosomal enI"mes
- <ynthesie! in ./ an! transporte! to the Lolgi
- 3o%e from cis to trans-Lolgi li"e secretory proteins
- ,ransport %esicles that !eli%er lysosomal enymes to en!olysosome bu! from the trans-Lolgi
25
networ"
- <election of lysosomal enymes !ue to the presence on them of a uni=ue mar"er: mannose-6-
p#osp#ate -a!!e! to 4-lin"e! oligosacchari!es in the cis-Lolgi
- Vey enyme : =lc)Ac p#osp#otransferase8 !eficient in $-cell !isease
-Llc42c phosphotransferase functions in the cis-Lolgi to a!! the 3CN tag to the lys# enyme
- Vnow to a!! the tag because of lysosomal RpatchS on enyme (!etails un"nown*
- /eceptor proteins cluster an! become concentrate! in clathrin-coate! %esicles in trans-Lolgi8
they bin! mannose-CN at pH T, release it at pH C: to transport lys# enymes, 3CN receptor
proteins in trans-Lolgi cluster an! bin! mCN at pH T, clathrin coate! %esicles form, lea%e trans
Lolgi, lose coat, mo%e to lysosome an! fuse with it, releasing 3CN tag at pH C
- 2fter lea%ing Lolgi, %esicles lose clathrin coats before fusing with en!osomes7en!olysosomes
- /eceptors recycle to trans-Lolgi8 not foun! in mature lysosomes: the receptors that brough the
enyme to the lysosome go bac" to the trans-Lolgi to pic" up more enyme
- /ecycling inhibite! by ammonia, chloro=uine
- $n some cells mannose-CN receptors also present on outsi!e of the cell surface, to reco%er
lysosomal enymes secrete! into e'tracellular me!ium
PER3<6S35ES
- 4K, part of $3 system, post translational import of proteins, all proteins ma!e in cytosol
- Narticles which contain most of cellsM Catalase Q one or more enymes which use molecular
o'ygen to remo%e hy!rogen atoms from specific substrates (pero.i$ase, 6-amino aci! o'i!ase,
urate o'i!ase*8 Fatalase uses hy!rogen pero'i!e pro!uce! to o'i!ie a %ariety of substrates
(phenols, formic aci!, formal!ehy!e, alcohol* or to pro!uce water Q K2 (safety !e%ice which
pre%ents accumulation of o'i!iing agent, hy!rogen pero'i!e*
- function ; hy!rogen pero'i!e !eto'ification, alchohol !eto'ification
- $n li%er o'i!ie about half of ethanol ingeste!
- Foun! in li%er, "i!ney, protooa, many cell types of higher plants, an! other cells (usually in
lower amounts*8 %ery !i%erse organelles, e%en in !ifferent cells of a single organism may contain
!ifferent sets of enymes
- $!entifie! by specific staining with the !iaminobeni!ine reaction for pero'i!ase acti%ity 9 loo"
i!entical to lysosomes, the only way to !istinguish is that !iaminobeni!ine !ar"er color
- ,ypical inclusion foun! in rat hepatic pero'isomes : the nucleoi!:
- Nero'isomes until recently thought to be forme! as !ilations of the /./8 it is now clear that new
2C
pero'isomes are only forme! from pre-e'isting ones by growth Q !i%ision (similar to mitochon!ria*
fission
- Lrow by upta"e of new pero'isomal proteins from cytosol
- 2ll components of pero'isomes importe! from the cytosol (amino aci! se=uence acts as a
specific signal*8 receptor proteins on membrane surface
- @ell*eger s"n$rome: !ue to !efect(s* in pero'isomal proteins import
E<3C9T3S6S
2 !istinct steps: bilayer a!herence an! bilayer Hoining (remember E leaflets, 2 bilayers*
- 3embrane fusion catalye! by fusogenic proteins
E.oc"tosis of secretor" proteins store$ in gran%les re2%ires,
1# secretagog%es 9 signals, interact with receptor sites locate! in the plasma membrane# ;
hormones, neurotransmitters (me!iate! by increase in calcium or c23N*
2# ligan!-receptor interaction trans!uce! into a pro'imal biochemical intracellular response:
mobiliation an! ele%ation of intracellular free Fa 2Qconcentration, or acti%ation of a!enylate
cyclase an! ele%ation of intracellular cyclic 23N (c23N* le%els#
- Falcium plays a critical role in regulating e'ocytosis in a number of systems#
- ,he o%erall process is in!epen!ent of protein synthesis, but re=uires metabolic energy 9 2,N
hy!rolysis
3# 3o%ement of the secretory granule from its site of formation7storage to the plasma membrane#
-sometimes in%ol%es microtubules, inhibite! by colchicines (inhibit microtubules*
E# Fusion of the secretory granules with the plasma membrane, an! !ischarge of the granule
content regulate! by specific enymes
- 6oc"ing ; %esicle comes to cell surface but still maintains membrane bilayer# Fusion ;
membrane bilayer is bro"en an! secretion occurs#
E)D3C9T3S6S
- Leneral term applie! to the upta"e by cells of particles by encirclement with cell processes
(p#agoc"tosis*, flui! in %acuoles forme! by in%agination of the surface membrane
(pinoc"tosis*, specific upta"e of e'tracellular proteins7pepti!es7other materials (receptor-
me$iate$ en$oc"tosis potoc"tosis*
Pinoc"tosis / 4LU6D
2T
- 5esicular upta"e of flui! containing low 30 solutes, soluble macromolecules, colloi!al particles
(too small to be %isualie! with the light microscope*, etc#
- Fan be !istinguishe! in two types :
- micropinocytosis (flui! ta"en up in minute in%aginations of the cell surface %isible only by ,.3*
-ca%eolin coats on ca%eolae (tiny in%agination* in%ol%e! in this 9 cells in culture use this to ta"e
up culture with nutrient in it, e'tensi%e form of fee!ing#
- macropinocytosis (un!ulating fol!s capture !roplets %isible with the phase-contrast microscope -
typically occurs at the thin peripheral portion of flattene! cells in culture to eat* (e%en less specific
than micro in that no "nown proteins are nee!e! for this* ?amellipo!ia (membrane fol!ings*
-4on specific in that you !o not nee! certain proteins to be able to pinocytie an! you grab
whate%er you grab 9 in%ol%es fol!ing of plasma membrane on itself#
Receptor-me$iate$ en$oc"tosis
- ,his process is highly specific (re=uires ligan!-receptor interaction* an! in%ol%es many
physiologically important molecules, happens in most tissues7cells
- <pecialie! regions of the surface membrane in%ol%e! : clathrin coate! pits - following
en!ocytosis, coate! %esicles forme!
- Foat compose! primarily of a single protein, clathrin (30 1B0,000* - minor proteins also
present specifically associate! with clathrin (a!aptins, etc# - see ne't ?ecture*
- Nolygonal networ" of clathrin, %isualie! by ,.3, <.3 (tris"elions*
- Gncoating 2,Nase (member of hspT0 family of stress response proteins* remo%es coat from
clathrin-coate! %esicles as soon as it enters cell
- ,here are also other Mcoate! %esiclesM in cells (not coate! with clathrin* in%ol%e! in intracellular
%esicular transport processes (e#g# among Lolgi cisternae - see ne't ?ecture*
- /ole of receptor-me!iate! en!ocytosis in cholesterol import - ?6? receptors: ?6? receptor Q
?6? brought into cell an! separate! in lysosome, ?6? release! into cytosol an! receptor sent
bac" to cell surface
- .n!ocytic %esicles !eli%er their contents to en!osomes (early an! then late en!osomes* which,
after fusion with Lolgi !eri%e! transport %esicles containing lysosomal enymes, become
en!olysosomes, an! finally lysosomes
- .arly en!osomes are important sorting centers, !eci!e some things shoul! cycle bac" to cell
surface but other components shoul! mo%e on#
- Potoc"tosis: se=uestration an! transport of small molecules by ca%eolae# <pecific receptors
in%ol%e!# 6on>t generate %esicles, ha%e en%aginations forming that are close! to outsi!e then
2B
reopen# Dest stu!ie! e'ample: the folate receptor
-6on>t nee! a folate gra!ient to bring folate in, will still transport it in
-Folate receptor on cell surface bin!s folate (?$L246*, ca%eolin causes tiny in%agination
(ca%eolae* an! folate is release! into cell
-no R%esicleS actually enters cell# ,he receptors concentrate folate in these little in%aginations
so when the channel opens you ha%e more folate there then in cytoplasm so it mo%es !own it>s
concentration gra!ient an! into the cell#
- Transc"tosis (transfer from one e'tracellular space to another in polarie! epithelial cells*
- $gL upta"e in newborn small intestine
- $g2 secretion in epithelial cells
P#agoc"tosis / LAR=E PART6CLES &!acteria'
$ngestion of particles large enough to be %isible with the light microscope# $n mammals
macrophages an! neutrophils %ery acti%e in phagocytosis#
1* 2ttachment by interaction of a ligan! on the particle with surface receptors on the membrane of
the phagocyte8
2* $ngestion : ipper-li"e process
- 2ttachment !oes not re=uire energy, ingestion !oes an! is temperature-!epen!ent
- ?arge, flat clathrin patches, similar to those characteristic of coate! pits an! %esicles (see
receptor-me!iate! en!ocytosis* obser%e! on the cytoplasmic si!e of phagosomes8
- Following internaliation, phagosomes fuse with lysosomes
7ES6CULAR TRA)SP3RT, SU55AR9
- <ignal pepti!es an! signal patches - me!iate at least some of the intracellular sorting
processes
1# import into nucleus (e#g# large ,, nucleoplasmin* nuclear import signals
2# import into ./ (signal pepti!es* <ignal Nepti!e, start an! stop transfer signals
3# retention in lumen of ./ (V6.?* V6.?;DiN an! VAAA;others, resi!ent ./ prot
E# import into mitochon!ria specific import signals (post-transl*
5# import into lysosomes 3CN tag, receptor proteins, etc#
C# en!ocytosis ligan!s, clathrin, other signals
-early en!osome ; sorting center, can mo%e stuff bac" to cell surface, to late en!osome, or
can become late en!osome
2W
- Constit%ti+e +s Reg%late$ secretor" pat#*a"s A SPEC646C6T9 34 C35PART5E)TS
- !ifferent types of coate! membrane regions7%esicles (clathrin, coatomer, ca%eolin*
Flathrin ; en!ocytotic %esicles an! for %esicles that bu! from trans golgi to generate %esicles
fille! with lysosomal enymes that fuse with lysosomes# <ame clatrhin, !ifferent lin"er proteins
FKN $ ; forwar! pathway from cis golgi trans an! then plasma membrane - re%erse pathway
golgi ./ Fop $$ ; ./ cis golgi networ"
- assembly - !isassembly of clathrin coats, a!aptins: same clathrin throughout, uses !ifferent
a!aptins in !ifferent %esicles !epen!ing what it>s transporting an! where it>s coming from
- coatomer-coate! %esicles - their role in intracellular %esicular transport (./-to-Lolgi8 cis-to-
trans Lolgi8 ,L4-to-surface membrane*8 T subunits (FKNs*, 2/F, monomeric - trimeric L
proteins
- 2/F (monomeric L,Nase, fatty aci! tail* regulates both assembly - !isassembly of coatomer
coats assembly: e'change L6N7L,N (catalye! by L4/N, inhibite! by Drefel!en 2*, fatty aci!
tail e'pose!, insertion into !onor membrane, recruitment of coatomer subunits, bu!!ing, pinching
off of %esicles !isassembly: !oc"ing at target membrane, L,Nase-acti%ating-protein, L,N
hy!rolysis, 2/F retracts tail, !isassembly of coat L,N ; tail, L6N ; no tail (can>t bin!*
- +-S)AREs t-S)AREs F Ra! proteins !etermine selecti%ity of %esicles !oc"ing
-proteins on %esicle surface recognie! by complementary proteins on target compartment
surface
- /ab proteins: L,Nases whose affinities for !ifferent ligan!s change when boun! to L6N or
L,N
6onor Fompartment
o Foat helps select what is incorporate! in a certain %esicle an! what is not#
o Formation of bu! in%ol%es aggregation of /ab-L,N that>s specific for !onor
compartment# L,N is essential for bin!ing of /ab to %esicle, ne!e! for uncoiling of
lipophyllic tail#
Din!ing to target compartment
o 3embrane fusion, /ab effector bin!s to /ab, L,N hy!rolye! an! /ab becomes
soluble not membrane boun! 9 !onor compartment has right L.F to ma"e
membrane soluble again (attach L,N cause e'posure of lipophyillic tail*
-when /ab-L,N is boun! to %esicle it has a lipi! tail which is recognie! by Ra! effector on
target membrane (in a!!ition to %-t <42/. interaction*
- after fusion, /an-L,N is hy!rolye! to remo%e from membrane (now part of target
30
membrane* an! sent bac" to !onor compartment (no more lipi! tail*
-L6N is replace! by L,N by L.F an! /an-L,N can be use! again in another %esicle
- fusion of %esicles to target membrane:
-content of %esicle becomes part of luminal comp# of target an! %esicle membrane components
become part of target membrane
-comple' process: )S4: cytosolic component that ai!s in !issociation of %-<42/. an! t-
<42/. (necessary to complete fusion* by hy!rolying 2,N an! pro%i!ing energy for !issoc)
- S)APs in%ol%e!
CELL SUR4ACE P3LAR6T9
- cell surface $omains (e#g# apical7basolateral*
- Fellular Hunctions are generally assume! to function as barriers
-,ight Hunction pre%ents proteins from mo%ing laterally from !omain to !omain
- LN$-anchore! proteins are foun! e'clusi%ely in the apical plasma membrane
Cell s%rface polarit" in Epit#elia
- .pithelial cells are organie! into sheets that separate compartments of the organism# ,he cells
in the epithelium are lin"e! through Hunctional comple'es so that they form a selecti%e
permeability barrier# .pithelial cells are specialie! to perform a wi!e %ariety of %ectorial
functions# ,ransporting epithelia, such as those of the renal tubule, absorpti%e epithelia such as
those of the intestine, an! secretory epithelial cells (e#g# hepatocytes* are typical e'amples of
epithelia that create an! maintain concentration gra!ients between the compartments they
separate# ,hese %ectorial functions reflect, an! !epen! on, the polar organiation of the epithelial
cells# .pithelial cells accomplish these functions by localiing !istinct sets of cell surface
components to separate regions of the plasma membrane# ,he basal aspect of the epithelial cell
layer usually rests on an e'tracellular matri', which is often organie! into a basement
membrane#
2pical an! basolateral !omains !iffer in both lipi! an! protein composition# ?ipi!s stu!ie! in
only a few cell types : in the absorpti%e %illus cells, glycolipi!s an! cholesterol are much more
abun!ant in the apical surface, phosphati!ylcholine in the basolateral !omain#
Nroteins stu!ie! in much more !etail, an! represent typical mar"ers of epithelial cell polarity#
For e'ample, sucrase, aminopepti!ase, lactase are only in the apical surface of the intestinal
%illus cells, 4aQ, VQ
-2,Nase in the basolateral membrane#
31
-Lolgi uses !ifferent types of %esicles to get stuff to apical an! basolateral membranes, mista"es
in this are fi'e! by en!osomes
<orting at le%el of transgolgi networ", some bu!s are !estine! for apical or lateral7basal
membrane#
Fomponents are mo%e! to lateral membrane, some stay because they belong there,
others are reta"en up by en!ocytosis an! en! up in early en!osomes# .arly en!osomes
are sorting centers an! mo%e bac" some component to lateral7basal membrane because
they belong there but mo%e some up to apical#
<orting !one in en!osome after insertion in lateral membrane#
Use of 7ir%ses in t#e st%$" of epit#elial cell polarit" : /o!rigue'-Doulan an! <abatini were
the first to !emonstrate that en%elope! %iruses bu! in a polarie! manner from infecte! 36FV
cells# ,his fin!ing pa%e! the way for the use of %iral glycoproteins as probes to stu!y the
biogenesis an! transport of apical an! basolateral proteins in epithelial cells in culture# Following
infection by en%elope! %iruses, host protein synthesis is suppresse! an! large =uantities of %iral
surface glycoproteins are synthesie!# ,his amplification facilitates the stu!ies of plasma
membrane biogenesis8 the assumption is ma!e that in infecte! cells the basic processes of
membrane protein synthesis an! intracellular transport are not altere!# $n the case of 36FV cells,
it was shown that the L protein of %esicular stomatitis %irus (5<5* is inserte! mainly into the
basolateral plasma membrane, whereas the spi"e glycoproteins of influena %irus beha%e as
apical plasmalemmal proteins#
,he <orting process of newly synthesie! membrane proteins !estine! to !ifferent parts of the
surface membrane is essentially still a mystery# 2pical an! basolateral proteins seem to remain
together !uring transport through the Lolgi comple'8 ,he trans-most part of the Lolgi comple' is
emerging as a goo! can!i!ate for the sorting process, an! the recently !escribe! ,rans-tubular
networ" continuous with the Lolgi apparatus may be an important part of the sorting machinery#

BioAP 3160 - LECTURE 13
Str%ct%re of 5itoc#on$ria
- 3itochon!rion boun!e! by smooth-contoure! outer membrane (about T nm thic"* an! an inner
membrane 8 two membranes separate! by a space (membrane space or intracristal space*
- 5itoc#on$rial 5atri. insi!e, gel-li"e#
32
- 3atri' granules: about spherical, osmiophilic inclusions# 642, boun! FaQQ, ribosomes
- /ibosomes an! 642 insi$e the mitochon!ria#
- 4umber of mitochon!ria7cell an! number of cristae7mitochon!rion relate! to energy re=uirement
- Fristae %ery %ariable in shape - sharp angulations, slen!er %illi, tubular cristae (steroi! secreting
cells*, branching tubules (protooan*, prismatic tubules (triangular in cross section*#
- Frystalline an! other inclusions fre=uently obser%e!#
- 4umber of mitochon!ria7cell %ery %ariable: li%er, B00-20008 oocytes of echino!erms, 150,0008
Liant amoeba Fhaos chaos, about half million# 3ay be ran!omly !isperse! in cytoplasm, or
concentrate! aroun! the cell center#
-3itochon!ria are %ery motile within the cell an! usually "now where to go (where 2,N nee!e!* 9
in "i!ney epithelium, lots of 4a7V pumps there so nee! a lot of 2,N
- 3itochon!ria pic" up a lot of calcium (calcium homeostasis 9 !ifferent than ./, calcium ta"en
up isn>t easy to release*# 2poptosis starts with release of cytochrome F in mitochon!ria
5ain f%nctions of mitoc#on$rial :s%!-compartments:
5atri.: - o'i!ation of pyru%ate, fatty aci!s
- citric aci! cycle
- 642 replication, protein synthesis (mitochon!rial t/42s, ribosomes*
6nner mem!rane : 5./@ highly selecti%e
- respiratory chain
- 2,N synthesis (2,N synthetase*
- specific carriers
6ntermem!rane space, - phosphorylation of nucleoti!es other than 2,N
3%ter mem!rane: - permeable to molecules less than 10,000 !altons (porin*
6solation an$ c#aracteriIation of 5itoc#on$rial mem!ranesH
- ?ipi! content: outer, E0:, inner, 20: - outer membrane contains more cholesterol, higher in
phosphati!yl inositol, lower in car!iolipin#
-$nner membrane has a ?K, of car!iolipin: a lipi! that ma"es the inner membrane super selecti%e
- 3itochon!rial enymes are highly compartmentalie!#
5itoc#on$rial D)A
- ?ocalie! in the matri' (multiple copies* - ,ypical properties:
1# Fircular molecule, 5-C Jm long, 15-1T "b pairs (in animals*
2# Duoyant !ensity in FsFl !iffers from that of nuclear 642
3# 4o histones
33
E# Fo!es for two r/42Ms, set of 22 t/42Ms (all mitochon!ria specific*, 13 protein-co!ing
se=uences (2,Nase, Fytochrome b comple', cytochrome o'i!ase comple'*
5 # ?ittle space for regulatory 642 se=uences, !ifferent genetic co!e genes tightly pac"e!)
C# 2ll other mitochon!rial proteins co!e! for by nuclear 642#
- 3itochon!rial ribosomes: smaller than cytoplasmic ribosomes, li"e bacterial ones
- S"m!iont #"pot#esis: 3itochon!ria an! Fhloroplasts are intracellular pro"aryotic parasites1
- 3itochon!rial genes are an e'ample of non-3en!elian inheritance, an! are maternally inherite!
-3at# inherite! bc maHority of cytoplasm in a ygote comes from egg, an! mitochon!rial 642 is
floating in cytoplasm ygotes get mostly maternal mitochon!rial material
S"nt#esis of 5itoc#on$rial Proteins an$ lipi$s
- ?ipi! components of mitochon!rial membranes importe! from ./ %ia carrier proteins
- 3ost of the proteins foun! in mitochon!ria are co!e! for in the cell nucleus, synthesie! on free
ribosomes, an! importe! into the mitochon!rion by specific processes# post-translationally
> $istinct $estinations , outer membrane, intermembrane space, inner membrane, matri' space
Translocation of 5atri. proteins
- proteins importe! from cytosol within few minutes from completion of synthesis
-usually boun! by chaperones once they>re synth# so they !on>t fol! before they get where they>re
going in the mitochon!ria# Nrecursor proteins for the mitochon!ria ha%e a signal se=uence,
mitochon!rial import se=uence that is recognie! by ,K3 comple'# 6ifference b7w matri' protein
an! inner membrane protein is e'istence of secon! signal se=uence that !irects it through the
inner membrane#
- they ha%e almost always a signal pepti!e (minimum about 12 amino aci!s*remo%e! by a signal
pepti!ase in matri'
- receptor proteins for signal pepti!e in outer mitochon!rial membrane 1
- import specifically at contact sites between outer an! inner membranes
- two main translocators: ,K3 an! ,$3 (2 types of ,$3*
- energ" re=uire! for import, 1 steps
1# ,K3: initial penetration of signal pepti!e: occurs at low temperature, re=uires 2,N hy!rolysis
2# ,$3: mo%ement of remaining protein chains into matri': re=uires electrochemical gra!ient
across mitochon!rial inner membrane
- unfol!ing of proteins for translocation8 chaperones (members of hspC0 family* an! 2,N re=uire!
3E
Protein transport into intermem!rane space an$ inner mem!rane
- First proteins transporte! into matri' an! first signal se=uence is clea%e!
- then secon!, %ery hy!rophobic amino aci! se=uence, unco%ere! by clea%age of signal pepti!e,
acts as a signal for reinsertion (Re'portS* into inner membrane
- inner membrane proteins remain inserte! into membrane, without clea%age of the secon! signal
se=uence
- if !estine! for intermem!rane space, steps abo%e are followe!, en!ing with the clea%age of the
secon! signal se=uence so that protein is free in intermembrane space
6nsertion of c"tosolic proteins into o%ter mem!rane
- 2,N hy!rolysis nee!e!, but not membrane potential - mechanism still unclear
Di+ision of mitoc#on$ria
1# D)A replication - synthesis of mitochon!rial ribosomes an! structural proteins1
2# Nair of membranes forms sept%m e'ten!ing across the organelle# 6istincti%e structure, not
Hust an e'tension of the cristae#
3# Fircumferential fol! of outer membrane in%a!es the space between septal membranes#
E# 0hen a!%ancing e!ges of fol! meet an! fuse, separation of !aughter mitochon!ria is
complete!#
56T3C(3)DR6AL B63E)ER=ET6CS - C(E563S53T6C T(E3R9
- 0hy is 2,N an energy source1 .=uilibrium 2,N 26N Q Ni
- 2,N concentration in the cytosol is "ept much higher than at e=uilibrium8 the ten$enc" of 2,N
to reach e=uilibrium, that is, the large !ecrease in free energ" associate! with 2,N hy!rolysis
un!er the con!itions normally present insi!e li%ing cells, is what !ri%es couple!, energetically
unfa%orable reactions 9 2,N has a ten!ency to hy!rolye, that ten!ency !ri%es r'ns#
- 3itochon!ria an! Fhloroplasts are primarily responsible for the rapi! con%ersion of 26N into
2,N, which maintains the cytosolic 2,N concentration much higher than at e=uilibrium#
- Llycolysis (substrate le%el o'i!ation* also pro!uces 2,N while con%erting glucose into pyru%ate
in the cytosol, but its 2,N yiel!7glucose is much lower than for o'i!ati%e phosphorylation#
6nsi$e 5itoc#on$ria &in 5atri.' o'i!ati%e metabolism fuele! largely by fatty aci! (!eri%e! from
the storage form, fat*, an! pyru%ate (pro!uce! from glucose, by glycolysis in cytosol*# <electi%ely
35
transporte! into mitochon!ria#
- fatty aci!s bro"en !own in the fatty aci! o'i!ation cycle to 2cetyl-Fo2
- pyru%ate also con%erte! to 2cetyl-Fo2
- 2cetyl-Fo2 enters Vrebs cycle an! generates 426H, F26H2 which then interact with the
respiratory chain
C(E563S53T6C T(E3R9
- 3itochon!rial inner membrane pro%i!es a framewor" for e- transport processes that con%ert
energy of 426H or F26H2 into 2,N#
- C#emiosmosis - lin" between chemical an! transport processes - e- are passe! along the
electron transport chain of proteins, embe!!e! in an ion impermeable membrane, an! the energy
release! in the e- passage is use! to pump protons form one si!e of the membrane (the matri'
si!e* to the other (the intermembrane space*# pH gra!ient is generate! (IT in matri', YT in the
intermembrane space* an! membrane potential (negati%e in matri', positi%e in intermembrane
space*#
- ,his generates an Electroc#emical Proton =ra$ient with a correspon!ing Proton 5oti+e
4orce -$n mitochon!ria of a typical cell, 4aQ7HQ e'change maintains pH in matri' relati%ely close
to neutrality (bring 4aQ into matri' to "eep pH from getting too high w7 loss of HQ*
- Nroton 3oti%e Force, in a!!ition to !ri%ing 2,N synthesis, also !ri%es %arious transport
processes &P3> infl%. Ca10 infl%. ATP-ADP antiport s"stem 92,N is sent out of
mitochon!ria into cell against its gra!ient' against the correspon!ing concentration gra!ients#
ATP-S"nt#etase &41-4o Comple.' - couples HQ flu' inwar! (!ri%en by Proton 5oti+e 4orce* to
2,N synthesis (from 26N Q Ni*# ?arge comple' (W polypepti!es* acts as a transmem!rane (0
c#annelH
- Nurifie! 2,N synthetase (without membrane* actually is an 2,Nase: hy!rolyses 2,N
-re%erse function stimulate! when cell senses that there>s too much 2,N
- $nner mitochon!rial membranes, without F1 fragment of 2,N synthetase, o'i!ie 426H in the
presence of K2, but no 2,N is synthesie! in the process (the portion of 2,N-synthetase
embe!!e! in the membrane, the Fo fragment, ma"es membrane permeable to HQ an! effecti%ely
!issipates the proton gra!ient*#
- F1 fragment, a!!e! bac" to strippe! inner mitochon!rial membranes, reconstitutes 2,N
synthesis#
3C
- .legant P!emonstrationP of Fhemiosmotic theory: artificial coupling, in synthetic liposomes
- Gn!er special con!itions, 2,N synthetase can be ma!e to function in re%erse: 2,N is
hy!rolye! with concomitant transport of HQ in re%erse#
- Dalance between free-energy change for mo%ing 2-3 HQ across membrane (proportional to
protonmoti%e force* an! free-energy change for 2,N synthesis in matri'#
- 0hen 2,N hy!rolye! in cytoplasm, ratio 2,N:26N in matri' falls, an! 2,N synthetase starts
again to ma"e 2,N# (when cell senses too little 2,N in cell, starts to ma"e more 2,N*
- HQ
63)3P(3RES uncouple o'i!ati%e phosphorylation by !issipating HQ gra!ient
- Bro*n 4at Cells energy of o'i!ation entirely !issipate! as heat: hibernating animals, human
babies# (HQ gra!ient uncouple! from 2,N synthesis so HQ Hust !iffuses bac" !own its gra!ient
an! heat is release!, but no 2,N ma!e*
RESP6RAT3R9 C(A6)
K%erall reaction: H2Q172 K2 ; H2K carrie! out in se%eral steps# H- (from 426H* ; HQ Q 2 e-
(boun! to 1st carrier* then e- flow through at least 15 !ifferent electron carriers8 in the process,
energy release! by e-use! to mo%e HQ#
-use step wise process because if Hust let e- go from 426H to K2 !irectly, woul! be so fa%orable
it woul! cause an e'plosion
- 3 5aGor Comple.es:
a* )AD( $e#"$rogenase comple. (at least 12 polypepti!es* transfers e- to ubi=uinone
b* !-c1 comple. (B polypepti!es*, probably present as !imer - recei%es e- from ubi=uinone an!
transfers them to cytochrome c
c* C"toc#rome 3.i$ase Comple. (T polypepti!es*, !imer - uses E e- from cytochrome c an! K2
to pro!uce 2'H2K
- 3 maHor comple'es, U!i2%inone (also calle! coenyme X* an! c"toc#rome c !iffuse in plane
of membrane as in!i%i!ual entities# 5ectorial organiation
-organie! in or!er, but not stuc" together, "in! of flowing laterally an! bump into each other
- Kn the a%erage, for each 426H o'i!ie!, 2e- transferre!, 32,N (ma'imum* synthesie!#
.- Flow:
426H426H !ehy!rogenaseubi=uinonecyt# b-c1 comple'cyt# ccyt# o'i# comple'K2
3T