BICD 110 Lecture 5 ER to Gogli

Rothman anatomy hypothesis

chemistry governs anatomy

Requirements of vesicle transport

1) components to attract coat
2) coat formation/coat proteins

3) coat disassociation

4) machinery for fusion

GAP

GTPase Activating Protein, stimulated GTP → GDP, taking GTPases from ON to OFF

GEF

Guanine Nucleotide Exchange Factor, swap GDP for GTP, turning GTPases from OFF to ON

COPII

anterograde transport, ER to ERGIC

COPI

Retrograde transport, ERGIC to ER

Proteins involved in COPII coats

Sar1-GDP, Sar1-GTP (GTPase)

Sec12 (ER membrane protein with GEF activity)

Sec23/24 (Inner coat, GAP activity)

Sec13/31 (outer coat)

COPII Formation Steps

1) Sar1-GDP interacts with Sec12 on membrane, conformational change of Sar1-GTP

2) Sar1-GTP recruits coat proteins Sec23/24 (inner) and Sec13/31 (outer)

3) Sec23 GAP activity cause Sar1-GTP → Sar1-GDP

4) Sar1-GDP disassociates from membrane, coat disassembles from vesicle

Anterograde signals

acidic, hydrophobic

DXE, EXE, DXD, FF, YY, FY

Sec23/24

Recognize CYTOPLASMIC signals (amino acid sequence) on CARGO membrane protein → implies that cargo proteins are transmembrane

lectin

carbohydrate binding protein

ERGIC-53

binds to high mannose N-glycans (from ER), type I transmembrane protein, dihydrophobic in cytoplasmic tail

neutral pH: glycans bound

acidic: sugars released (at ERGIC)

types of SNARE proteins

v-SNARE (vesicle SNARE, comes from donor membrane)

t-SNARE (target SNARE, comes from receiver membrane)

GOLGINS

create coccon around Golgi, prevent access to Golgi/ERGIC

NSF ATPase

disassembles 4-helix bundle

4-helix bundle

complex that that allows for fusion of vesicle to target membrane, 1 v-SNARE + 3 t-SNARE

Proteins Involved in COPI Coats

1) Arf1-GDP/ GTP (GTPase)

2) GBF1 (GEF activity, changes GDP to GTP in Arf1)

3) coatomer (binds signals in cytosolic cargo protein domains)

4) ArfGAP (Arf1-GTP → GDP)

COPI Formation Steps

1) Arf-GDP interacts with membrane proteins p23/24

2) GBF-1 GEF activity: Arf-GDP —> GTP, conformational change that causes hydrophobic end of Arf-GTP to insert into the membrane

3) Arf-GTP recruits coat protein complex (7 different proteins) coatomer, which binds specific sequences in membrane cargo protein

4) ArfGAP activity stimulates hydrolysis of Arf-GTP to Arf-GDP

5) Arf-GDP dissociates from membrane and causes disassembly of coat

Retrograde Transport Signals

Basic:

KKXX
K/HDEL

RR

RXR
RRR

KDEL Receptor

recognizes KDEL sequence which indicates that it’s misrouted ER membrane that needs to go back to the ER

Retrieval of ER luminal proteins

1) luminal protein accidentally incorporated in COPII vesicle

2) vesicle fuses with ERGIC, KDEL receptors in ERGIC → acidic pH promotes binding of receptor to KDEL signal sequence on ER luminal protein

3) Binding of ER luminal protein to KDEL causes it to be incorporated into a COPI vesicle

4) COPI goes back to ER, fuses. Higher pH of ER lumen promotes dissociation of KDEL protein (ER protein) from receptor