CHAPTER 15

INTRACELLULAR COMPARTMENTS
AND TRANSPORT
2004 Garland Science Puli!"in#
Me$rane%Encl&!ed Or#anelle!
15-1 Name the membrane-bounded compartments in a eucaryotic cell where each of the
functions listed below takes place.
A. Photosynthesis
B. Transcription
C. Oxidatie phosphorylation
!. "odification of secreted proteins
#. $teroid hormone synthesis
%. !e&radation of worn-out or&anelles
'. New membrane synthesis
(. Breakdown of lipids and toxic molecules
24'
15-2 )abel the structures of the cell indicated by the lines on the fi&ure below*
%i&ure +,--.
A. nucleus
B. free ribosomes
C. rou&h endoplasmic reticulum
!. 'ol&i apparatus
#. cytosol
%. endosome
'. plasma membrane
(. lysosome
/. mitochondrion
0. peroxisome
15-3 1ou discoer a fun&us that contains a stran&e star-shaped or&anelle not found in any other
eucaryotic cell you hae seen. On further inesti&ation you find the followin&
,. the or&anelle possesses a small &enome in its interior.
.. the or&anelle is surrounded by two membranes.
2. esicles do not pinch off the or&anelle membrane.
3. the interior of the or&anelle contains proteins similar to those of many bacteria.
-. the interior of the or&anelle contains ribosomes.
(ow mi&ht this or&anelle hae arisen4
24(
Pr&)ein S&r)in#
15-4 %or each of the followin& sentences5 fill in the blanks with the best word or phrase
selected from the list below. Not all words or phrases will be used6 each word or phrase
should be used only once.
Plasma membrane proteins are inserted into the membrane in the
777777777777777777. The address information for protein sortin& in a
eucaryotic cell is contained in the 777777777777777777 of the proteins.
Proteins enter the nucleus in their 777777777777777777 form. Proteins
that remain in the cytosol do not contain a 777777777777777777.
Proteins are transported into the 'ol&i apparatus ia
777777777777777777. The proteins transported into the endoplasmic
reticulum by 777777777777777777 are in their 777777777777777777
form.
amino acid se8uence 'ol&i apparatus sortin& si&nal
endoplasmic reticulum plasma membrane transport esicles
folded protein translocators unfolded
15-5 9hat would happen in each of the followin& cases4 Assume in each case that the protein
inoled is a soluble protein5 not a membrane protein.
A. 1ou add a si&nal se8uence :for the #;< to the amino-terminal end of a normally
cytosolic protein.
B. 1ou chan&e the hydrophobic amino acids in an #; si&nal se8uence into char&ed
amino acids.
C. 1ou chan&e the hydrophobic amino acids in an #; si&nal se8uence into other5
hydrophobic5 amino acids.
!. 1ou moe the amino-terminal #; si&nal se8uence to the carboxyl-terminal end of
the protein.
24*
15-6 1ou are tryin& to identify the peroxisome-tar&etin& se8uence in the thiolase en=yme from
yeast. The thiolase en=yme normally resides in the peroxisome and therefore must
contain amino acid se8uences that are used to tar&et the en=yme for import into the
peroxisome. To identify the tar&etin& se8uences5 you create a set of hybrid &enes that
encode fusion proteins containin& part of the thiolase protein fused to another protein5
histidinol dehydro&enase :(!(<. (!( is a cytosolic en=yme re8uired for the synthesis
of the amino acid histidine and cannot function if it is locali=ed in the peroxisome. 1ou
&enetically en&ineer a series of yeast cells to express these fusion proteins instead of their
own ersions of these en=ymes. /f the fusion proteins are imported into the peroxisome5
the (!( portion of the protein cannot function and the yeast cells cannot &row on media
lackin& histidine. 1ou obtain the followin& results*
%i&ure +,-->
9hat re&ion of the thiolase protein contains the peroxisomal tar&etin& se8uence4 #xplain
your answer.
250
15-7 9hat is the role of the nuclear locali=ation se8uence in a nuclear protein4
:a< /t is bound by cytoplasmic proteins that direct the nuclear protein to the nuclear
pore.
:b< /t is a hydrophobic se8uence that enables the protein to enter the nuclear
membranes.
:c< /t aids protein unfoldin& in order for the protein to thread throu&h nuclear pores.
:d< /t preents the protein diffusin& out of the nucleus ia nuclear pores.
15-8 A &ene re&ulatory protein5 A5 contains a typical nuclear locali=ation si&nal but
surprisin&ly is usually found in the cytosol of cells. 9hen the cell is exposed to
hormones5 protein A moes from the cytosol into the nucleus where it turns on &enes
inoled in cell diision. 9hen you purify protein A from cells that hae not been treated
with hormones5 you find that protein B is always complexed with it. To determine the
function of protein B5 you en&ineer cells lackin& the &ene for protein B. 1ou compare
normal and defectie cells by usin& differential centrifu&ation to separate the nuclear
fraction from the cytoplasmic fraction and then separate the proteins in these fractions by
&el electrophoresis. 1ou identify the presence of protein A and protein B by lookin& for
their characteristic bands on the &el. The &el you run is shown below*
%i&ure +,--?
On the basis of these results5 what is the function of protein B4 #xplain your conclusion
and propose a mechanism for how protein B works.
15-9 9hich of the followin& statements about import of proteins into mitochondria are T;@#4
:a< The si&nal se8uences on mitochondrial proteins are usually carboxyl terminal.
:b< The first sta&e of import of a mitochondrial protein is across the outer membrane
into the intermembrane space.
:c< "ost mitochondrial proteins are not imported from the cytosol but are synthesi=ed
inside the mitochondria.
:d< "itochondrial proteins are translocated across the inner and outer membranes
simultaneously.
:e< "itochondrial proteins cross the membrane in their natie5 folded state.
251
15-10 Proteins destined to enter the endoplasmic reticulum
:a< are transported across the membrane after their synthesis is complete.
:b< are synthesi=ed on free ribosomes in the cytosol.
:c< be&in to cross the membrane while still bein& synthesi=ed.
:d< cross the membrane in a folded state.
:e< all remain within the endoplasmic reticulum.
15-11 After isolatin& the rou&h endoplasmic reticulum from the rest of the cytoplasm5 you
purify the ;NAs attached to it. 9hich of the followin& proteins do you expect the ;NA
from the rou&h endoplasmic reticulum to encode4
:a< $oluble secreted proteins
:b< #; membrane proteins
:c< "itochondrial membrane proteins
:d< Plasma membrane proteins
:e< ;ibosomal proteins
15-12 Briefly describe the mechanism by which the presence of an internal stop-transfer
se8uence in a protein causes the protein to become embedded in the lipid bilayer as a
transmembrane protein with a sin&le membrane-spannin& re&ion. Assume that the protein
has an amino terminal si&nal se8uence and Aust one internal hydrophobic stop-transfer
se8uence.
15-13 @sin& &enetic en&ineerin& techni8ues5 you hae created a set of proteins that contain two
:and only two< conflictin& si&nal se8uences that specify different compartments. Predict
which si&nal would win out for the followin& combinations. #xplain your answers.
A. $i&nals for import into the nucleus and import into the #;.
B. $i&nals for export from the nucleus and import into the mitochondria.
C. $i&nals for import into mitochondria and retention in the #;.
15-14 A protein traerses the plasma membrane three times in the orientation shown below
:N B amino terminus5 C B carboxyl terminus6 the hydrophobic membrane-spannin&
re&ions are shown as open boxes<. This protein is known to hae a si&nal se8uence that
is cleaed by si&nal peptidase in the #;.
%i&ure +,--,3
$ketch the #; membrane and the arran&ement of the newly synthesi=ed protein chain
after it has completed its entry into the #; membrane but before any action of si&nal
peptidase. Be sure to label the cytosol5 the #; lumen5 the si&nal se8uence5 and the amino
and carboxyl termini of the protein in your dia&ram.
252
15-15 The fi&ure below shows the orientation of a multipass transmembrane protein after it has
completed its entry into the #; membrane :part A< and after it &ets deliered to the
plasma membrane :part B<. This protein has an amino-terminal si&nal se8uence :depicted
as the dark &rey membrane spannin& box<5 which is cleaed off in the endoplasmic
reticulum by si&nal peptidase. The other membrane-spannin& domains in the protein are
depicted as open boxes. 'ien that any hydrophobic membrane-spannin& domain can act
as either a start-transfer or a stop-transfer re&ion5 draw the final conse8uences of the
actions described below on the orientation of the protein in the plasma membrane. Be
sure to indicate on your drawin& the extracellular space5 the cytosolic face5 and the
plasma membrane5 as well as the amino- and carboxyl-termini of the protein.
%i&ure +,--,-
A. !eletin& the first si&nal se8uence.
B. Chan&in& the hydrophobic amino acids in the first5 cleaed5 se8uence to char&ed
amino acids.
C. Chan&in& the hydrophobic residues in eery other transmembrane se8uence to
char&ed residues5 startin& with the first5 cleaed5 si&nal se8uence.
15-16 #xamine the multipass transmembrane protein shown in %i&ure +,--,>. 9hat would
you predict would be the effect of conertin& the first hydrophobic transmembrane
se&ment to a hydrophilic se&ment4 $ketch the arran&ement of the modified protein in the
#; membrane.
%i&ure +,--,>
25+
,e!icular Tran!-&r)
15-17 %or each of the followin& sentences5 fill in the blanks with the best word or phrase
selected from the list below. Not all words or phrases will be used6 each word or phrase
should be used only once.
Proteins are transported out of a cell ia the 777777777777777777 or
777777777777777777 pathway. %luids and macromolecules are
transported into the cell ia the 777777777777777777 pathway. All
proteins bein& transported out of the cell pass throu&h the
777777777777777777 and the 777777777777777777. Transport esicles
link or&anelles of the 777777777777777777 system. The formation of
777777777777777777 in the endoplasmic reticulum stabili=es protein
structure.
carbohydrate 'ol&i apparatus
disulfide bonds hydro&en bonds
endocytic ionic bonds
endomembrane lysosome
endoplasmic reticulum protein
endosome secretory
exocytic
15-18 Name two functions of the protein coat of esicles that bud from membranous or&anelles
used in esicular transport.
15-19 An indiidual transport esicle
:a< contains only one type of protein in its lumen.
:b< will fuse with only one type of membrane.
:c< is endocytic if it is traelin& toward the plasma membrane.
:d< is enclosed by a membrane with the same lipid and protein composition as the
membrane of the donor or&anelle.
254
15-20 /n class we hae discussed how -$NA;#s and t-$NA;#$ mediate the reco&nition of a
esicle with its tar&et membrane so that a esicle displayin& a particular type of -
$NA;# will only fuse with a tar&et membrane containin& a complementary type of t-
$NA;#. /t is also known that in some cases5 -$NA;#s and t-$NA;#s may also
mediate fusion of identical membranes. /n yeast cells5 ri&ht before the formation of a
new cell5 esicles deried from the acuole will come to&ether and fuse to form a new
acuole destined for the new cell. @nlike the situation weCe discussed in class5 the
acuolar esicles contain both -$NA;#s and t-$NA;#s. 1our friend is tryin& to
understand the role of these $NA;#s in the formation of the new acuole and wants to
consult with you re&ardin& the interpretation of his data.
1our friend has desi&ned an in&enious assay for the fusion of acuolar esicles utili=in&
alkaline phosphatase. The protein alkaline phosphatase is made in a DproE form that
must be cleaed in order for the protein to be actie. 1our friend has desi&ned two
different strains of yeast* strain A produces the DproE form of alkaline phosphatase :pro-
Pase<5 while strain B produces the protease that can cleae pro-Pase into the actie form
:Pase<. Neither strain has the actie form of the alkaline phosphatase5 but when acuolar
esicles from the strains A and B are mixed5 fusion of esicles &enerates actie alkaline
phosphates5 whose actiity can be measured and 8uantified.
%i&ure +,--.FA
1our friend has taken each of these yeast strains and further en&ineered them so that they
express only the -$NA;#s5 the t-$NA;#s5 both :the normal situation<5 or neither
$NA;#. (e then isolates acuolar esicles from all strains and tests the ability of each
ariant form of strain A to fuse with each ariant form of strain B5 usin& the alkaline
phosphatase assay. The data are shown in the &raph depicted in %i&ure +,--.FB. On this
&raph5 the $NA;# present on the esicle of the particular yeast strain is indicated as DE
:for the presence of the -$NA;#< and DtE :for the presence of the t-$NA;#<.
255
%i&ure +,--.F B
9hat does his data say about the re8uirements for -$NA;#s and t-$NA;#s in the
acuolar esicles4 Be sure to comment on whether it is important to hae a specific type
of $NA;# :that is5 - or t-$NA;#< on each esicle.
Secre)&r. Pa)"/a.
15-21 N-linked oli&osaccharides on secreted &lycoproteins are attached to
:a< nitro&en atoms in the polypeptide backbone.
:b< the serine or threonine in the se8uence Asn-G-$erHThr.
:c< the amino terminus of the protein.
:d< the aspara&ine in the se8uence Asn-G-$erHThr.
:e< the aspartic acid in the se8uence Asp-G-$erHThr.
15-22 Name two types of protein modification that can occur in the #; but not in the cytosol.
15-23 /f you were to remoe the #;-retention si&nal from a protein that normally resides in the
#; lumen5 where do you expect the protein will ultimately end up4 Be sure to explain
your reasonin&5 for full credit.
250
15-24 "atch the set of labels below with the numbered label lines on %i&ure ,--.3.
%i&ure +,--.3
A. Cisterna
B. 'ol&i stack
C. $ecretory esicle
!. trans 'ol&i network
#. cis 'ol&i network
25'
15-25 A plasma membrane protein carries an oli&osaccharide containin& mannose :"an<5
&alactose :'al<5 sialic acid :$A<5 and N-acetyl&lucosamine :'lcNAc<. These su&ars are
added to the protein as it proceeds throu&h the secretory pathway. %irst5 a core
oli&osaccharide containin& "an and 'lcNAc is added5 followed by 'al5 "an5 $A5 and
'lcNAc in a particular order. #ach addition is cataly=ed by a different transferase actin&
at a different sta&e as the protein proceeds throu&h the secretory pathway. 1ou hae
isolated mutants defectie for each of the transferases5 purified the membrane protein
from each of the mutants5 and identified which su&ars are present in each mutant protein.
The results are summari=ed in Table +,--.-.
Table +,--.-
$u&ars present in the purified protein
Cell lackin&* "an 'al $A 'lcNAc
A. Oli&osaccharide I I I I
protein transferase
B. 'alactose J I I J
transferase
C. $A transferase J J I J
!. 'lcNAc J I I less than in
transferase normal cells
%rom these results5 match each of the transferases :A5 B5 C5 !< to its subcellular location
selected from the list below. :Assume that each location contains only one en=yme.<
,. Central 'ol&i cisternae
.. cis 'ol&i network
2. #;
3. trans 'ol&i network
15-26 %or each of the followin& sentences5 choose one of the options enclosed in s8uare
brackets to make a correct statement.
New plasma membrane reaches the plasma membrane by the
Kre&ulatedHconstitutieL exocytosis pathway. New plasma membrane
proteins reach the plasma membrane by the Kre&ulatedHconstitutieL
exocytosis pathway. /nsulin is secreted from pancreatic cells by the
Kre&ulatedHconstitutieL exocytosis pathway. The interior of the trans
'ol&i network is KacidicHalkalineL. Proteins that are constitutiely secreted
Ka&&re&ateHdo not a&&re&ateL in the trans 'ol&i network.
15-27 /n a cell capable of re&ulated secretion5 what are the three main classes of proteins that
must be separated before they leae the trans 'ol&i network4
25(
End&c.)ic Pa)"/a.!
15-28 Name three possible fates for an endocytosed molecule that has reached the endosome.
15-29 %or each of the followin& sentences5 fill in the blanks with the best word or phrase
selected from the list below. Not all words or phrases will be used6 each word or phrase
should be used only once.
#ucaryotic cells are continually takin& up materials from the extracellular
space by the process of endocytosis. One type of endocytosis is
7777777777777777775 which inoles utili=in& 777777777777777777
proteins to form small esicles containin& fluids and molecules. After
these esicles pinch off from the plasma membrane5 they will fuse with the
7777777777777777775 where the uptaken materials are sorted. A second
type of endocytosis is 7777777777777777775 which is used to take up
lar&e esicles that can contain microor&anisms and cellular debris.
"acropha&es are especially suited for this process5 as they extend
777777777777777777 :sheetlike proAections of their plasma membrane< to
surround the inadin& microor&anisms.
chaperone 'ol&i apparatus pseudopods
cholesterol mycobacterium rou&h #;
clathrin pha&ocytosis $NA;#
endosome pinocytosis transcytosis
25*
15-30 %ibroblast cells from patients 95 G5 15 and M5 who each hae a different inherited defect5
all contain Dinclusion bodies5E which are lysosomes filled with undi&ested material. 1ou
wish to identify the cellular basis of these defects. The possibilities are*
,. a defect in one of the lysosomal hydrolases.
.. a defect in the phosphotransferase that is re8uired for mannose->-phosphate
ta&&in& of the lysosomal hydrolases.
2. a defect in the mannose->-phosphate receptor5 which binds mannose->-phosphate
ta&&ed lysosomal proteins in the trans 'ol&i network and deliers them to
lysosomes.
1ou find that when some of these mutant fibroblasts are incubated in media in which
normal cells hae been &rown5 the inclusion bodies disappear. This leads you to suspect
that lysosomal hydrolases are bein& secreted by the constitutie exocytic pathway in
normal cells and are bein& taken up by the mutant cells. :/t is known that some mannose-
>-phosphate receptor molecules are found in the plasma membrane and can take up and
delier lysosomal proteins ia the endocytic pathway.< 1ou incubate cells from each
patient with media from normal cells and media from each of the other mutant cell
cultures5 and &et the followin& results.
"edia
%rom %rom %rom %rom %rom
normal cultures cultures cultures cultures
cells of 9 cells of G cells of 1 cells of M cells
Cell )ine
Normal J J J J J
9 I I I I I
G J J I I I
1 J J I I J
M J J I J I
J indicates that the cells appear normal6 I indicates that the cells still hae inclusion
bodies.
%or each patient :95 G5 15 M< indicate which of the defects :,5 .5 2< they are most likely to
hae.
15-31 (ow is it that the low p( of lysosomes protects the rest of the cell from lysosomal
en=ymes in case the lysosome breaks4
200
H&/ 1e 2n&/3 Trac4in# Pr&)ein and ,e!icle Tran!-&r)
15-32 1ou hae created a '%P fusion to a protein that is normally secreted from yeast cells.
$ince you hae learned about the use of temperature-sensitie mutations in yeast to study
protein and esicle transport5 you obtain a collection of three mutant yeast strains5 each
one defectie in some aspect of the protein secretory process. Bein& a &ood scientist5 you
of course5 also obtain a wild-type control strain. 1ou decide to examine the fate of your
'%P fusion protein in these arious yeast strains and en&ineer the mutant strains to
express your '%P fusion protein. (oweer5 in your excitement to do the experiment5 you
reali=e that you did not label any of the mutant yeast strains and no lon&er know which
strain is defectie in what process. 1ou end up numberin& your strains with the numbers
, throu&h 35 and then you carry out the experiment anyway5 obtainin& the followin&
results :note that the black dots represent your '%P fusion protein<*
%i&ure +,--2.
Name the process defectie in each of these strains. ;emember that one of these strains
is your wild-type control.
201
An!/er!
15-1 A Photosynthesis B chloroplast
B. Transcription B nucleus
C. Oxidatie phosphorylation B mitochondrion
!. "odification of secreted proteins B 'ol&i apparatus and rou&h endoplasmic
reticulum :#;<
#. $teroid hormone synthesis B smooth #;
%. !e&radation of worn-out or&anelles B lysosome
'. New membrane synthesis B #;
(. Breakdown of lipids and toxic molecules B peroxisome
15.2 $ee %i&ure A,--..
%i&ure A,--.
15-3 A &enome5 a double membrane5 ribosomes5 and proteins similar to those found in bacteria
are eidence for an or&anelle hain& eoled from an en&ulfed bacterium.
15-4 Plasma membrane proteins are inserted into the membrane in the endoplasmic
reticulum. The address information for protein sortin& in a eucaryotic cell is contained
in the amino acid sequence of the proteins. Proteins enter the nucleus in their folded
form. Proteins that remain in the cytosol do not contain a sorting signal. Proteins are
transported into the 'ol&i apparatus ia transport vesicles. The proteins transported into
the endoplasmic reticulum by protein translocators are in their unfolded form.
202
15-5 A. The protein will now be transported into the #; lumen.
B. The altered si&nal se8uence will not be reco&ni=ed and the protein will remain in
the cytosol.
C. The protein will still be deliered into the #;. /t is the distribution of hydrophobic
amino acids that is important5 not the actual se8uence.
!. The protein will not enter the #;. Because the carboxyl terminus of the protein is
the last part to be made5 the ribosomes synthesi=in& this protein will not be
reco&ni=ed by the $;P and carried to the #;.
15-6 The peroxisomal tar&etin& se8uence lies between amino acids number ,FF and number
,.-. Any fusion protein containin& this se8uence can be tar&eted for import into the
peroxisome :because the yeast cannot &row on media lackin& histidine< while the fusion
proteins lackin& this re&ion do not tar&et the fusion protein for import into the
peroxisome :because the yeast do &row on media lackin& histidine<. The most important
pieces of data are the fusion proteins containin& amino acids ,FFI.FF of the thiolase
protein fused to (!( and the fusion protein containin& amino acids ,I,.- of the thiolase
protein fused to (!(. Both of these fusion proteins do not allow &rowth on media
lackin& histidine and can be used to define the minimal re&ion necessary for tar&etin&
thiolase for import into the peroxisome.
:Note that althou&h these experiments show that amino acids ,FFI,.- are necessary5
these experiments do not show that this re&ion is sufficient for peroxisomal tar&etin&. /t
is possible that amino acids ,FFI,.- is sufficient5 or5 it could be that this re&ion
collaborates with redundant si&nals between amino acids ,I,FF or ,.-I.FF.<
15-7 :a<
15-8 The data on the &el shows that protein A is always found in the nucleus in the absence of
protein B. Therefore5 any mechanism that is proposed must explain this result.
On possible answer is that protein B binds protein A and masks the nuclear locali=ation
si&nal. /n the presence of hormone5 protein B interacts with the hormone5 which chan&es
its conformation so that it can no lon&er bind protein A. 9hen protein B no lon&er binds
to protein A5 the nuclear locali=ation si&nal on protein A is now exposed and protein A
can enter the nucleus. Therefore5 in the absence of protein B5 the nuclear locali=ation
si&nal on protein A is always exposed and protein A resides in the nucleus.
Another possible answer is that protein B binds protein A and se8uesters it by keepin&
protein A in some subcellular compartment5 away from the nucleus. /n the presence of
hormone5 protein B interacts with the hormone5 chan&in& its conformation so that it can
no lon&er bind to protein A. 9hen protein B is not present5 protein A can enter the
nucleus in the presence or absence of hormone.
15-9 :d<
15-10 :c<
20+
15-11 :a<5 :b<5 and :d< The rou&h #; consists of #; membranes and polyribosomes that are in
the process of translatin& and translocatin& proteins into the #; membrane and
lumen. Thus all proteins that end up in the lysosome5 'ol&i apparatus5 or plasma
membrane5 or are secreted5 will be encoded by the ;NAs associated with the
rou&h #;. "itochondrial and ribosomal proteins are translated on free cytosolic
ribosomes.
15-12 The amino-terminal si&nal se8uence initiates translocation and the protein chain starts to
thread throu&h the translocation channel. 9hen the stop-transfer se8uence enters the
translocation channel5 the channel dischar&es both the si&nal se8uence and the stop-
transfer se8uence sideways into the lipid bilayer. The si&nal se8uence is then cleaed5 so
that the protein remains held in the membrane by the hydrophobic stop-transfer se8uence.
15-13 A. The protein would enter the #;. The si&nal for a protein to enter the #; is
reco&ni=ed as the protein is bein& synthesi=ed and will end up either in the #; or
on the #; membrane. Proteins destined for the nucleus &et reco&ni=ed by
cytosolic nuclear transport proteins once they are fully synthesi=ed and fully
folded.
B. The protein would enter in the mitochondria. /n order for a nuclear export si&nal
to work5 the protein would hae to end up in the nucleus first and thus would need
a nuclear import si&nal for the nuclear export si&nal to &et utili=ed.
C. The protein would enter the mitochondria. /n order to be retained in the #;5 the
protein needs to enter the #;. $ince there is no si&nal for #; import5 the #;
retention si&nal would not function.
15-14 The N-terminal si&nal se8uence initiates translocation of the protein across the #;
membrane. The si&nal se8uence will be cleaed off by si&nal peptidase5 leain& the
amino-terminus of the protein in the luminal side of the #; membrane. @pon fusion to
the plasma membrane5 the amino terminus of the protein will reside in the extracellular
space.
%i&ure A,--,3
204
15-15 A. !eletin& the first si&nal se8uence completely would conert the next membrane-
spannin& domain into an internal start-transfer si&nal and would inert the
orientation of the protein :see %i&ure A,--,-A<.
B. Chan&in& the hydrophobic amino acids to char&ed amino acids destroys the
ability of the se8uence both to act as a si&nal se8uence and to become a
membrane-spannin& se8uence. Therefore5 the adAacent membrane spannin&
domain will now become an internal start-transfer se8uence and the protein will
be inerted5 as seen aboe in part A. The mutated si&nal se8uence would not &et
cleaed off5 since it would remain on the cytoplasmic side of the membrane and
si&nal peptidase is found only inside the #; :see %i&ure A,--,-B<.
C. "utatin& eery other membrane spannin& re&ion so that they are now char&ed
:and thus cannot span the membrane< would decrease the number of
transmembrane re&ions and increase the si=e of the internal loops between
membrane-spannin& re&ions :see %i&ure ,--,-C<.
%i&ure A,--,-
15-16 As shown in %i&ure A,--,>5 elimination of the first transmembrane se&ment :by makin&
it hydrophilic< would be expected to reerse the orientation of the protein in the
membrane. 9hat ori&inally was the second transmembrane se&ment :a stop-transfer
si&nal<5 would now be read as a start-transfer si&nal and would hae the opposite
orientation in the membraneNas would all the remainin& transmembrane se&ments.
Althou&h the N-terminus would still be in the #; lumen5 all the rest of the external parts
of the protein would swap positions so that what was in the cytosol would now be in the
#; lumen5 and ice ersa.
%i&ure A,--,>
205
15-17 Proteins are transported out of a cell ia the secretor or e!octic pathway. %luid and
macromolecules are transported into the cell ia the endoctic pathway. All proteins
bein& transported out of the cell pass throu&h the endoplasmic reticulum and the "olgi
apparatus. Transport esicles link or&anelles of the endomem#rane system. The
formation of disulfide #onds in the endoplasmic reticulum stabili=es protein structure.
15-18 ,. The proteins in the coat help shape the membrane into a bud.
.. The proteins in the coat can also select car&oes for transport.
15-19 Choice :b< is the correct answer. An indiidual esicle may contain more than one type of
protein in its lumen :choice :a<<5 all of which will contain the same sortin& si&nal :or will
lack specific sortin& si&nals<. #ndocytic esicles :choice :c<< &enerally moe away from
the plasma membrane. The esicle membrane will not necessarily contain the same lipid
and protein composition as the donor or&anelle5 since the esicle is formed from a
selected subset of the or&anelle membrane from which it budded :choice :d<<.
15-20 /n order to &et maximal leels of acuolar esicle fusion5 esicles from each strain must
carry both -$NA;#s and t-$NA;#$. #xperiment ,5 which represents the normal
scenario5 is the only experiment where ,FFO alkaline phosphatase actiity is measured.
(oweer5 as lon& as complementary $NA;#s are present on the esicles5 some esicle
fusion does occur :see experiments 25 35 >5 P5 ?5 Q<. /f both esicles are missin& either -
$NA;#s :experiment .< or t-$NA;#s :experiment -< or both $NA;#s :experiment ,F
and ,,<5 the leel of fusion is ery low. /t does not matter whether a t- or -$NA;# is on
the esicle of a particular strain5 as lon& as the esicle from the other strain contains a
complementary $NA;# :compare experiments 2 and 35 > and P5 and ? and Q<.
15-21 :d<
15-22 ,. Proteins in the #; can under&o disulfide bond formation. :This does not occur in
the cytosol because of its reducin& enironment.<
.. Proteins in the #; can under&o &lycosylation. :'lycosylatin& en=ymes are not
found in the cytosol.<
:$i&nal-se8uence cleaa&e is also an acceptable answer5 althou&h not really what
this 8uestion is referrin& to.<
15-23 The protein would end up in the extracellular space. Normally5 the protein would &o
from the #; to the 'ol&i apparatus5 &et captured because of its #;-retrieal si&nal5 and
return to the #;. (oweer5 without the #;-retrieal si&nal5 the protein would eade
capture5 ultimately leae the 'ol&i ia the default pathway5 and become secreted into the
extracellular space. The protein would not be retained anywhere else alon& the secretory
pathway5 as it presumably has no si&nals to promote such locali=ation since it normally
resides in the #; lumen.
15-24 AN26 BN,6 CN-6 !N36 #N.
200
15-25 AN2 :oli&osaccharide protein transferase B #;<
BN, :&alactose transferase B central 'ol&i cisternae<
CN3 :$A transferase B trans 'ol&i network<
!N. :'lcNAc transferase B cis 'ol&i network
Proteins are modified in a stepwise fashion in the 'ol&i apparatus5 with early steps takin&
place in the cis 'ol&i5 intermediate steps takin& place in the central 'ol&i cisternae5 and
late steps occurrin& in the trans 'ol&i network. /f each en=yme produces the substrate for
the next step5 then a mutant lackin& the en=yme that cataly=es the addition of the first
su&ar will be missin& all of the su&ars5 a mutant lackin& the en=yme that cataly=es the
addition of the second su&ar will contain the first su&ar but will lack the other three5 and
so on. By this lo&ic5 mannose and 'lcNAc must be the first su&ars added5 additional
'lcNAc the second5 &alactose the third5 and $A the last. (ence5 the oli&osaccharide
protein transferase must be in the #;5 the 'lcNAc transferase in the cis 'ol&i5 the
&alactose transferase in the central 'ol&i5 and the $A transferase in the trans 'ol&i.
15-26 New plasma membrane reaches the plasma membrane by the constitutive exocytosis
pathway. New plasma membrane proteins reach the plasma membrane by the
constitutive exocytosis pathway. /nsulin is secreted from pancreatic cells by the
regulated exocytosis pathway. The interior of the trans 'ol&i network is acidic.
Proteins that are constitutiely secreted do not aggregate in the trans 'ol&i network.
15-27 The three main classes of proteins that must be sorted before they leae the trans 'ol&i
network in a cell capable of re&ulated secretion are :,< those destined for lysosomes5 :.<
those destined for secretory esicles5 and :2< those destined for immediate deliery to the
cell surface.
15-28 ,. recycled to the ori&inal membrane
.. destroyed in the lysosome
2. transcytosed across the cell to a different membrane.
15-29 #ucaryotic cells are continually takin& up materials from the extracellular space by the
process of endocytosis. One type of endocytosis is pinoctosis5 which inoles utili=in&
clat$rin proteins to form small esicles containin& fluids and molecules. After these
esicles pinch off from the plasma membrane5 they will fuse with the endosome5 where
the uptaken materials are sorted. A second type of endocytosis is p$agoctosis5 which is
used to take up lar&e esicles that can contain microor&anisms and cellular debris.
"acropha&es are especially suited for this process5 as they extend pseudopods :sheetlike
proAections of their plasma membrane< to surround the inadin& microor&anisms.
20'
15-30 9N2 :defect in mannose->-phosphate receptor<
GN. :defect in phosphotransferase<
1N,6 MN, :defect in lysosomal hydrolases<6 these will be defects in two different
lysosomal acid hydrolases.
A cell that has no mannose->-phosphate receptor will be able to make all the lysosomal
hydrolases properly5 but will not be able to send them to the lysosome and will also not
be able to scaen&e hydrolases from the external media. (ence5 this cell line cannot be
rescued by culture media that has had lysosomal hydrolases secreted into it and thus will
not be rescued by any of the media tested here. A cell line that has no phosphotransferase
will be able to scaen&e hydrolases from the external medium5 but since all of the cellCs
own hydrolases will lack the mannose->-phosphate ta&5 it will be rescued only by media
from a cell line that is able to make all of the hydrolases. Cell lines missin& one hydrolase
will be rescued by media from any cell line that is able to secrete that hydrolase in a
mannose->-phosphate ta&&ed form6 in addition5 media from cultures of cells missin& a
hydrolase will rescue any cell line with another type of defect.
15-31 The lysosomal en=ymes are all acid hydrolases5 which hae optimal actiity at the low
p( :about -.F< found in the interior of lysosomes. /f a lysosome were to break5 the acid
hydrolases would find themseles at p( P..5 the p( of the cytosol5 and would therefore
do little dama&e to cellular constituents.
15-32 $train A has protein accumulatin& in the #;5 which means that this cell has a mutation
that blocks transport from the #; to the 'ol&i apparatus. $train B has secreted protein5
and therefore is your wild-type control. $train C has protein accumulatin& in the 'ol&i
apparatus5 and thus has a mutation that blocks exit of proteins from the 'ol&i apparatus.
$train ! has protein accumulatin& in the cis-'ol&i network5 and thus has a mutation that
blocks the trael of proteins throu&h the 'ol&i cisternae.
20(