Endoplasmic reticulum: protein synthesis and modification,lipid synthesis

All eukaryotic cells have an ER, more than half the total membrane ER lumen or ER cisternal: one continuous space 10% of the total cell volume Protein and lipid synthesis: Both transmembrane and water soluble proteins are synthesized and translocated across the ER membrane

Free and membrane-bound ribosome

Rough and smooth ER can be separated by centrifugation

Smooth microsomes come from smooth ER, plasma membrane, Golgi apparatus, endosomes and mitochondria (exception: liver),

The signal hypothesis (1970s)

In vitro translation with or without microsomes: size protease ER signal sequence signal peptidase

Signal recognition particle

ER signal sequences and SRP direct ribosomes to the ER membrane

Sec61 complex at protein translocator

Signal sequence is removed from most

soluble proteins after translocation

Asparagine-linked (N-linked) precursor oligosaccharide

Oligossaccharyl transferase

Precursor oligosaccharide 14 sugars

Most proteins in the ER are added to the asparagine-linked (N-linked) precursor oligosaccharide

Synthesis starts in the cytosol and ends in lumen

Synthesis starts in the cytosol and ends in lumen Lipid intermediate is flipped across the bilayer by a transporter Oligosacchides are used as tags to mark the state of protein folding

Improperly folded proteins are exported from the ER and degraded in the cytosol

Unfolded protein response

Phospholipids are synthesized on the cytosolic leaflet of the ER

Biosynthesis of Phospholipids
Lipid synthesis symmetric an asymmetric

Vesicular trafficking from ER to Golgi

Pathways: Endocytic illustrated in green arrows Biosyntheticsecretory illustrated with red arrows Retrieval illustrated with blue arrows

Use of Different coats in vesicular traffic

Adaptin binds to cargo receptor and clathrin triskelion

Dynamin is a GTPase

Dynamin pinches off the bud

ATPase is required to remove the clathrin coat

Coat recruitment GTPases

ARF proteins: COPI & Clathrin Sar1 protein: COPII

GTP causes Sar1 to bind to membrane

GTPase works like a timer and causes disassembly shortly after the budding is complete

Rab effector proteins ensure specificity Snare mediate membrane fusion

Rab effector proteins (green) interact via Rab-GTP (yellow) for tethering vesicle and membrane Rab-GEF activate Rab proteins on both vesicle and membrane Snares complex responsible for docking synaptic vesicles

Recruitment of cargo molecules to ER transport vesicle

ER exit site (no ribosomes)

Selective process

ER retrieval signals: KKXX in ER membranes proteins KDEL sequence in soluble ER resident proteins

Cis

Trans

pH controls affinity of KDEL receptors- higher acidic on trans end Membrane proteins in Golgi and ER have shorter TM domains

Oligosaccharide processing in ER and Golgi

Two models of through Golgi

Supporting evidence: Collegen rods Scales on algae

Sample Midterm BICD 110 Cell Biology

1). The following modification occurs on nuclear lamins, which cause nuclear lamina to disassemble and nuclear envelope to be redistributed during cell cycle: a. N-linked glycosylation b. O-linked glycosylation c. Phosphorylation d. Myristalation 2). The function of the phospholipid exchange (transfer) proteins is: a. Flipping phospholipids from one leaflet to another b. Catalyzing glycosylation of phospholipids c. Moving newly synthesized phospholipids from smooth ER to other membrane compartments d. Transfer lipids into cell nucleus

2. True or false (10 questions) (20%)

________1).Lampbrush chromosomes are mitotic chromosomes.

________2).Proteins take up most of a cell dry weight. s T

3. Filling the blanks (10 questions) (20%)

1). The 30 nm fiber, found in interphase chromosomes can be unfolded a structure called beads on a string ____________________________ iw nucleosome core particles and DNA; th 2). The most common secondary structure of the transmembrane domains of membraneAlpha ________; spanning proteins is ______helix

4. Experiments (two experiments) (10%)

1) As shown in the diagram below, when a mouse cell and a human cell are induced to fuse into one hybrid cell, the membrane proteins from the two different cells were found intermixed with each other all over the fused cell.

a)

What was the main conclusion of this classical experiment? Proteins lateral diffusion b) Please briefly describe another experiment that can provide more quantitative measurement of this feature of membranes and membrane proteins that the above-mentioned experiment reveals Photobleaching- by photobleaching a small region of the plasma membrane the rate at which the protein heads laterally diffuse and mix with the rest of the membrane can be measured

5. Name the structures (two questions) (10%) 1). Please name the three important structures on eukaryotic chromosomes indicated as 1, 2 and 3.

telemere Replication of orgin

centromere

6. Short answers (two questions) (20%)

The following diagram shows protein translocation into a mitochondrian

a. Explain where energy is introduced to allow directed transport. Energy is introduced at TOM (cytosol) and TIM23 (mito) where ATP hydrolysis is used to remove Hsp70 chaperones that are binding protein b. Indicate where the TOM complex and the TIM23 complex are located. TOM complex outer MB where protein is translocated, TIM23 on inner MB