Protein trafficking (cytosol > ER > return to ER)

ER (structure)

branched tubules and flattened sacs enclose central lumen

• rough = membrane w/ bound ribosomes

• smooth = membrane w/o bound ribosomes

rough ER (function)

site of protein translation, folding, shipment to Golgi

*co-translational translocation

smooth ER (function)

lipid biosynthesis, Ca2+ storage, maintains cholesterol levels, makes enzymes that make lipoproteins (hepatocytes → LDL/HDL)

A protein with what signal sequence will be trafficked to the rough ER?

a cluster of 8+ nonpolar aa near N terminal (ER import)

Ex. Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala

What types of proteins have the ER import signal?

resident transmembrane and soluble ER proteins

*signal sequence does not make it a permanent resident of ER (i.e. soluble could leave/be secreted in another organelle)

What happens to the ER import signal after the polypeptide enters the organelle?

signal is cleaved by a peptidase

Where should a protein w/ a KDEL sequence @ the C-terminal end up?

the lumen of the rough ER

Will KDEL ever be cleaved?

no; this allows the protein to stay in the lumen and carry out translation, folding, shipment to golgi)

Mutated KDEL

will still be imported (signal not affected); would not stay in the organelle → transported to golgi and packaged/sent to extracellular space

Which proteins undergo co-translational translocation?

soluble, single-pass transmembrane, multi-pass transmembrane

*tail-anchored is post

What signal sequences would you find in PDI during translation (no cleaving yet)?

ER import sequence (8+ nonpolar aa), KDEL sequence

*soluble ER resident (in COP I vesicles)

Would PDI have any stop transfer sequences?

no, b/c it is meant to stay in the ER (soluble)

Tail-anchored proteins (mechanism)

energy = ATP

hydrophobic alpha-helix signal sequence (@ C-terminal)

no stop transfer

Get1/2 receptors

Get3, pre-targeting proteins

Transmembrane proteins (mechanism)

energy = hydrolysis

import signal sequence (@ N-terminal)

1+ stop transfer

SRP receptor

peptidase, protein, translocator proteins

N-linked glycolysations

oligosaccharides are added to the NH2 Asn side chains of the target protein (Asn-X-Ser or Asn-X-Thr; X is any aa except Pro)

• transferase facilitates addition (ER lumen)

*chem rxns only take place if active site is in right compartment

Why do soluble resident cytosolic proteins not have N-linked glycosylations?

b/c translation occurs in the cytosol and glycosylation enzymes are typically found on the luminal face of the rough ER

Why do soluble resident cytosolic proteins not have disulfide bonds?

b/c PDI, the enzyme that forms disulfide bonds is an ER resident → cytosolic proteins can’t reach it

How is glycosylation helpful for moderating protein folding in the ER?

chaperones bind to glycosylated regions → prevent formation of aggregates/correct misfolding

*timers that measure how long a protein remains unfolded

How does the cell prevent a protein from cycling through the process too many times?

a slow trimming of mannose off of the oligosaccharide → allows a new structure to be recognized by the lectin on the retro translocator to be exported out

What happens if the protein is prevented from re-entering the folding cycle?

it binds to lectin to be sent to the retro translocator → ubiquitinated and degraded in the proteasome

What is the purpose of the unfolded protein response?

maintain balance of protein folding in ER/buffers ER stress

• prevent aggregates from forming

What are some things the cell does during this response to alleviate the burden?

1) splice mRNA that codes for transcription of regulatory proteins

2) reduce protein translation, decrease selective concentration in ER

3) regulate proteolysis in Golgi

Where are fully processed proteins in the ER sent?

packaged into transport vesicles and sent to the cis golgi

What happens to proteins that aren’t folded correctly?

• the chaperones (lectin) will bind and hold onto unfolded proteins → prevent packaging

• misfolded proteins will have a conformation that hides the sorting signal

ER proteins (pH)

golgi: lower pH → higher [H+], promotes binding of KDEL to receptors packaged into COP I

ER: higher pH → lower [H+], promotes dissociation of KDEL from receptor

What would happen if the pH in the ER matched the Golgi?

the KDEL would stay bound to soluble proteins (no dissociation) → dysfunction in ER

Mitochondrial proteins

do not contain disulfide bonds

COP I

golgi → ER

COP II

ER → golgi

Clathrin

plasma membrane → endosomes

Where would a membrane transport protein w/ a large segment of non-polar aa in the N-terminal mutated to polar aa reside?

the protein will be localized to the cytosol