vesicular transport: early secretory pathway

Vesicular transport in the early secretory pathway

What cellular pathway is the focus of this lecture

Early secretory pathway

Transport from ER to Golgi and within the Golgi complex

What small GTPase initiates COPII vesicle formation

Sar1

What protein recruits Sar1 to the ER membrane

Sec12

What is the role of Sec12

GEF that exchanges GDP for GTP on Sar1

What happens to Sar1 after GTP binding

Sar1 inserts into the ER membrane

What structural feature allows Sar1 to insert into membranes

N-terminal amphipathic alpha helix

What coat is recruited by Sar1-GTP

COPII coat

What is the function of COPII vesicles

Forward transport from ER to Golgi

What experimental approach was used to study Sar1 function

Liposome reconstitution experiments

What happens when Sar1-GTP is added to liposomes

Induction of membrane curvature and tubulation

What happens when Sar1-GDP is added to liposomes

No significant membrane curvature

What does tubule formation in liposomes indicate

Induction of membrane curvature

Why were NTA liposomes used in Sar1 experiments

To artificially recruit His-tagged Sar1 to membranes

What tag was used to bind Sar1 to NTA liposomes

His epitope tag

Why is artificial recruitment important in these experiments

To separate membrane binding from curvature induction

What happens when Sar1 lacking the N-terminal helix is recruited to membranes

It fails to induce membrane curvature

What conclusion was drawn about the Sar1 N-terminal helix

It is required for membrane curvature

What happens when wild-type Sar1 and COPII are added to liposomes

Formation of free coated vesicles

What happens when mutant Sar1 lacking the N-terminus and COPII are added

Budding occurs but few vesicles are released

What does this show about vesicle scission

Combined curvature from Sar1 and COPII is required

Which coat protein was initially thought to mediate forward Golgi transport

COPI

Why was COPI function initially misinterpreted

Preconception that vesicles must mediate forward transport

How is Golgi structure visualised experimentally

Electron microscopy tilt series

Where are vesicles enriched on the Golgi complex

At the rims of Golgi cisternae

What experiment first demonstrated the secretory pathway

Pulse–chase autoradiography

Which scientist performed these experiments

George Palade

What cell type was used in Palade’s experiments

Pancreatic cells

What did early pulse–chase time points label

Endoplasmic reticulum

Where did labelled proteins accumulate at intermediate times

Golgi complex

Where did labelled proteins accumulate at late times

Secretory granules

What modification is used as a biochemical readout of Golgi transport

N-linked glycosylation

Where is the high-mannose oligosaccharide added

Endoplasmic reticulum

What modification occurs in the cis and medial Golgi

Addition of N-acetylglucosamine

What modification occurs in the trans Golgi

Addition of galactose and sialic acid

How can glycosylation changes be detected

SDS-PAGE molecular weight shifts

What enzyme sensitivity distinguishes ER from Golgi forms

Endo H sensitivity

What does Endo H resistance indicate

Passage through the medial Golgi

Why are Golgi enzymes useful markers

They are spatially polarised across cisternae

Why are enveloped viruses useful cargo models

They exploit host secretory pathways

Name a commonly used viral cargo protein

VSV G protein

What advantage do viruses provide experimentally

High expression and strong antibody availability

Who developed the in vitro intra-Golgi transport assay

Rothman

What was the key biochemical readout in Rothman’s assay

Incorporation of radioactive N-acetylglucosamine

What did donor Golgi membranes lack

GlcNAc transferase

What did acceptor Golgi membranes provide

GlcNAc transferase

What components were required for in vitro transport

ATP and cytosol

What non-hydrolysable GTP analogue was used

GTPγS

What effect does GTPγS have on Golgi transport

Vesicle accumulation without fusion

What did GTPγS reveal about GTP hydrolysis

It is required for vesicle consumption

What coat was purified from accumulated vesicles

COPI coat