Cell Biology Chapter 11 wip

endoplasmic reticulum (ER)

network of membrane-enclosed tubules and sacs (cisternae), extending from nuclear membrane throughout cytoplasm

-continuous membrane, largest organelle, 2 domains with different functions

-protein processing

protein processing: pulse-chase experiments

used to demonstrate the secretory pathway

-newly synthesized proteins were labeled with radioisotopes → location of radiolabeled proteins determined by autoradiography → incubation with non-labeled amino acids for different lengths of time

secretory pathway

rough ER → golgi → secretory vesicles → cell exterior

protein sorting

initial sorting occurs while translation is in progress

-free ribosomes in cytosol, membrane-bound ribosomes

free ribosomes in cytosol

inside nucleus, mitochondria, chloroplasts, peroxisomes

membrane-bound ribosomes

nuclear membrane, peroxisome membrane, golgi (plasma membrane, endosomes, lysosomes)

ribosomes targeted to the ER by

signal sequence

signal sequence

-~20 amino acids (including stretch of hydrophobic residues)

-usually located at amino terminus of polypeptide chain; signal removed when growing polypeptide chain enters ER

-cotranslational targeting of secretory proteins

cotranslational targeting of secretory proteins

SRP (signal recognition particle) binds to signal sequence (pauses translation) → brings ribosome to ER → binds to SRP receptor → SRP releases polypeptide → translation continues → polypeptide threaded through channel & into lumen of ER → signal peptidase cleaves signal sequence → translation continues until stop codon

insertion of proteins into the membrane

-transported along secretory pathway (membrane components, not soluble proteins)

-ER lumen is topologically equivalent to exterior of cell

-regions of polypeptide chains translocated into ER lumen correspond to domains of membrane proteins that are exposed on the cell surface

insertion of membrane proteins with internal transmembrane sequences

-internal transmembrane sequence recognized by SRP, but not cleaved by signal peptidase

-transmembrane region exits translocon laterally; polypeptides can be oriented in either direction across membrane

-N-terminus stays in cytosol, some occasionally leave C-terminus

insertion of a membrane protein with a cleavable signal sequence & an internal transmembrane sequence

after signal is cleaved, encounters transmembrane sequence in translocon, translation continues, polypeptide exits translocon

insertion of a protein that spans the membrane multiple times

second transmembrane stays in locon, repeat

post translation insertion of a protein with a C-terminal transmembrane sequence

targeting factor TRC40 brings protein to GET1/GET2 receptor in ER membrane

protein folding & processing

can occur either during translocation across the ER membrane or in the ER lumen

-HSP70 chaperone BiP binds to unfolded polypeptides as they cross the ER membrane (facilitates folding & assembly of multisubunit proteins)

-N-linked glycosylation

N-linked glycosylation

occurs in ER while translation still in progress

-oligosaccharide is synthesized on a lipid carrier

-glycosylphosphatidylinositol (GPI) anchors attach some proteins to PM (assembled in ER membrane & added to carboxy terminus of some polypeptides; exposed to outside of cell)

quality control in the ER

-protein folding is slow and inefficient, many are misfolded; chaperones and protein-processing enzymes act as protein-folding sensors

-ER-associated degradation

ER-associated degradation (ERAD)

misfolded proteins are identified, returned to cytosol, & rapidly degraded by ubiquitin-proteasome system

glycoprotein folding by calnexin (chaperone)

-recognizes partially folded glycoprotein, assists in completion of folding

-protein-folding sensor checks folding

—if correct, continues to golgi

—if incorrect, protein is targeted back to cytosol through ubiquitin-ligase to be degraded

unfolded protein response (UPR)

signaling pathway activated when an excess of unfolded proteins accumulates

-expansion of the ER and production of more chaperones

-if folding cannot be adjusted to a normal level, the cell undergoes programmed cell death

ER receptors activated in UPR

-IRE1 cleaves XBP1 pre-mRNA → XBP1 translated → TF activates transcription of UPR genes

-ATF6 cleaved to release active ATF6 (UPR TF)

-PERK phosphorylates eIF2 (inhibits general translation) & activates ATF$ (UPR TF)