D103 ER Protein Sorting (ALS 10, Videos 17 and 18)

what is the entry point into the secretory pathway

the ER

ER signal sequence and receptor

SRP (signal recognition particle as sig sequence) = binds its substrate via a hydrophobic pocket, which can accomodate N-terminal hydrophobic sorting signals and hydrophobic domains inside a protein 

• hydrophobic region at N term 

SRP receptor 

how are ribosomes directed to the ER membrane

via co-translational ER import; no chaperones

1. hydrophobic signal sequence on N term associates with SRP on the ribosome; moving along the mRNA

2. SRP receptor GTPase on the ER membrane associates with SRP bound to ribosome

3. when bound, translocon opens to allow protein thru ER membrane; GTP hydrolysis to inactivate srp receptor

4. signal peptidase cleaves signal sequence to release protein into er

synthesis of type I membrane protein

type I: single TMD; C-term is in cytosol = N-term in ER lumen

N term signal sequence: directs the protein for import into the ER

subsequent hydrophobic domain: TMD 

type ii protein

single tmd

c-term is in ER lumen = N term in cytosol

if + charged AA precede the domain, N-term is in the cytosol

what determines membrane protein topology

its insertion into the ER

• if no N term signal sequence is present, hydrophobic domains can serve as internal signal sequence

synthesis of a multi-spanning membrane protein 

synthesized in ER 

1. start transfer sequence associates with binding site

2. continues moving thru the binding site until the stop transfer sequence is reaches 

3. mature double-pass TM protein in ER membrane 

challenges for the translocon

the translocon has to:

• form a pore to translocate proteins across the mem

• recognize hydrophobic domains

• release TMDs into the membrane

• be impermeable to ions

post translational ER membrane insertion

1. ribosome synthesizes the protein with signal sequence

2. chaperones keep it unfolded in the cytosol

3. binds to the Sec translocons → releases the chaperones

4. BiP in the ER lumen binds to translocon to release protein into lumen and cleave sig seq

ER import overview

sorting sig: hydrophobic at N-term; SRP

receptor: SRP receptor

translocation: ER translocon

energy: N/A; driven by polypeptide creation 

folding state: unfolded bc entering as being translated 

cleavage: yes; at membrane 

proteasome and ER

1/3 of the proteome is imported into the ER, then trafficked to organelles of the secretory pathway or secreted out

ER functions

• surrounded by lipid bilayer

• determines site of mito fission

• extends throughout the cell and this spread-out orgnization depends on kinesin-mediated transport

• ER stores Ca2+ (SR) that stimulated muscle contraction

ER domains 

rough ER: sheet-like

• major site of protein import into ER 

• entry point of the secretory pathway 

• protien topology is determiend

smooth ER: tubular

• synthesis of cholesterol and hormones

• phospholipid synthesis 

• Ca2+ storage 

• vesicle formation 

• contact sites with other organelles (mitos, endosomes) 

similarities between smooth and rough ER

• protein folding

• quality control

• stress response

er contact sites

1. lipid synthesis and exchange via ER-mitochondria interaction

2. endosome fission via ER-mito interaction

ER as a folding environment 

1. sugar tree on lipid gets trasnfered to the growing lipid via N-glycosylation; BiP and lectin are chaperones for folding 

2. form disulfide bonds with PDI 

3. arrange the peptide bond between proline and other AAs by peptide prolyl isomerase (PPI) 

4. membrane insertion as a membrane-spanning alpha helix 

5. protein oligomerization (form luminal alpha helix) 

N-glycosylation

initiated in the ER

Asn-X-Ser/Thr (consensus sequence)

• initiated in the ER

• continued in the Golgi

• motif is necessary but not sufficient

O-linked glycosylation

Ser/Thr

occurs only in the Golgi

role of glycosylation

protective layer at cell surface 

quality control 

lysosomal sorting (M6P) 

ER and disulfide bond formation

ER as site for disulfide bond formation (oxidative environment → promotes disulfide bond formation)

1. formation of disulfide bond: reduced substrate protein binds to oxidized PDI; PDI gets reduced and the oxidized substrate protein conencts

2. rearragement of disulfide bonds: protein with incorrect disulfide bonds reattach to reduced PDI, then pdi releases once correctly organized

chaperones and er

evaluate and modify the quality of the proteins

calnexin 

membrane-bound lectin (=sugar-binding proteins) with chaperone activity

calreticulin

soluble “relative” of calnexin with the same function

chaperones when glucosidase removes glucose

membrane-bound chaperone (calnexin) binds to sugar tree with 1 Glc

chaperones when glucosidase removes another glucose

if folded correctly: exit from ER 

if not: binds to soluble chaperone, Glc residue is added again 

purpose of new chaperone cycle

“buys” time for correct folding

protein folding problems

• trapped in misfolded conform

• mutation that leads to misfolding

• unassembled multimer subunits

what happens if a protein cannot be folded properly 

1. glucosidases (trim glucose residues from N-linked glycans)

2. glucosyltransferases (folding sensor for newly synthesized N-linked glycoproteins)

3. misfolded proteins bind and sequester BiP

4. unfolded protein response pathway 

unfolding response

first line of defense against er stress

1. IRE1: first sensor (homodimer, kinase)

• ribonuclease domain cleaves mRNA and iniates translation of transcription reg protein 1

2. PERK

• phosphorylation inactivates translation initiation factor → reduction of proteins entering the er

• selective translation of transcription reg porotein 2

3. ATF6

• regulated proteolysis in golgy releases transcription reg protein 3

results in activation of genes to increase protein-folding cap of er

ER-associated degradation (ERAD)

a cell’s repsonse to misfolded proteins in the ER

1. recog of malfolded protein

2. export from ER (pulling by ATPase)

3. poly-ubiquination (E3 ligase)

4. deglycosylation (N-glycanase)

5. degradation (proteasome)

ubiquitin 

conserved small protein (76 amino acids) 

• attached to lysines on target protein (no consensus) → poly-ubiquitination

• polyubiquitination leads to proteasome-dependent degradation 

ER folding overview

er chaps help the protein fold (BiP), form disulfide bridges (disulfide isomerase), and peptidul prolyl bonds (peptidylprolylisomerase)