vesicles

What pathway is the focus of the lecture

Vesicular transport in the early secretory pathway

What stages are included in the early secretory pathway

Transport from the ER to the Golgi and transport within the Golgi

What small GTPase initiates COPII vesicle formation

Sar1

What protein recruits Sar1 to the ER membrane

Sec12

What is the function of Sec12

Acts as a GEF to exchange GDP for GTP on Sar1

What happens to Sar1 after GTP binding

It inserts into the ER membrane

What structural feature of Sar1 mediates membrane insertion

N-terminal amphipathic alpha helix

Which coat complex is recruited by Sar1-GTP

COPII

What is the main role of COPII vesicles

Forward transport from ER to Golgi

What experimental system was used to study Sar1 activity

Liposome reconstitution experiments

What effect does Sar1-GTP have on liposomes

It induces membrane curvature and tubulation

What effect does Sar1-GDP have on liposomes

It does not induce membrane curvature

What does tubulation of liposomes indicate

Induction of membrane curvature

Why were NTA liposomes used in Sar1 experiments

To artificially recruit His-tagged Sar1 to membranes

Why is artificial recruitment important

To separate membrane binding from curvature induction

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

It binds but fails to induce 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 combined with liposomes

Free COPII-coated vesicles are formed

What happens when mutant Sar1 lacking the N-terminus is combined with COPII

Budding occurs but vesicle scission is inefficient

What does this demonstrate about vesicle formation

Combined curvature from Sar1 and COPII is required for scission

Which coat was initially assumed to mediate forward Golgi transport

COPI

Why was COPI function initially misinterpreted

Due to the preconception that vesicles mediate forward transport

How is Golgi structure visualised experimentally

Electron microscopy tilt series

Where are vesicles most abundant on the Golgi complex

At the rims of Golgi cisternae

What classic experiment revealed the secretory pathway

Pulse–chase autoradiography

Who performed the foundational secretory pathway experiments

George Palade

Which cells were used in Palade’s experiments

Pancreatic cells

Where are newly synthesised proteins first detected

Endoplasmic reticulum

Where do labelled proteins move after leaving the ER

Golgi complex

Where do labelled proteins accumulate at late time points

Secretory granules

What biochemical modification is used to track Golgi transport

O-linked glycosylation by addition of N-acetylglucosamine/glcNAc

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 experimentally

SDS-PAGE molecular weight shifts

What enzyme sensitivity distinguishes ER and Golgi forms of glycoproteins

Endo H sensitivity

What does Endo H resistance indicate

Passage through the medial Golgi

Why are Golgi enzymes useful transport markers

They are polarised across Golgi cisternae

Why are enveloped viruses useful model cargo

They exploit host secretory pathways

Name a commonly used viral cargo protein

VSV G protein

What experimental advantage do viruses provide

High protein expression and strong antibody availability

Who developed the in vitro intra-Golgi transport assay

Rothman

What was the biochemical readout of intra-Golgi transport

Incorporation of radioactive N-acetylglucosamine

What was lacking in the donor Golgi membranes

GlcNAc transferase

What did the acceptor Golgi membranes provide

GlcNAc transferase activity

What components were required for in vitro Golgi transport

ATP and cytosol

What non-hydrolysable GTP analogue was used in COPI studies

GTPγS

What effect does GTPγS have on Golgi transport

Vesicles accumulate but are not consumed

What does vesicle accumulation indicate

GTP hydrolysis is required for vesicle fusion or uncoating

Which coat complex was purified from accumulated vesicles

COPI

How many major subunits make up the COPI coat

Seven

What small GTPase recruits the COPI coat

ARF1

What membrane modification distinguishes ARF1 from Sar1

Myristoylation

What is the role of ARF1 myristoylation

Stabilises membrane association

What cargo motifs are recognised by COPI

Dilysine and RxR motifs

What is the function of the RxR motif

ER retention of unassembled protein complexes

What proteins stimulate COPI coat disassembly

ARF-GAPs

What activates ARF-GAPs

Membrane curvature and coat conformational changes

What unexpected components were enriched in COPI vesicles

Golgi glycosylation enzymes

What was not enriched in COPI vesicles

Forward secretory cargo

What is the primary function of COPI vesicles

Retrograde transport

In which direction does COPI-mediated transport occur

Golgi to ER and intra-Golgi recycling

What defect is seen in COPI mutants

ER proteins leak into the Golgi

What model explains Golgi transport without forward vesicles

Cisternal maturation

What happens to cisternae during cisternal maturation

They mature from cis to medial to trans

How are Golgi enzymes redistributed during cisternal maturation

Recycled backward via COPI vesicles

Why does cisternal maturation allow transport of large cargo

Large cargo cannot fit into small transport vesicles

What is membrane fusion

The merging of a transport vesicle membrane with a target membrane to allow cargo delivery

What are the two main challenges in membrane fusion

Specificity of fusion and overcoming the energetic barrier

What is the major energetic barrier to membrane fusion

The hydration barrier caused by water bound to charged polar lipid head groups

How close must membranes come to overcome the hydration barrier

Approximately 1 nanometer

Why does membrane fusion require proteins

Lipids alone cannot overcome the hydration and lipid tail energy barriers

What two biological systems illustrate membrane fusion

Transport vesicles in cells and enveloped viruses

Why are viral fusion proteins useful models

They show how protein conformational energy can drive membrane fusion

Which influenza protein mediates membrane fusion

Hemagglutinin

Which HIV protein directly mediates membrane fusion

gp41

What triggers conformational change in viral fusion proteins

Binding to host cell receptors

What is a fusion peptide

A hydrophobic peptide that inserts into the target membrane during fusion

Why is the fusion peptide compared to a transmembrane domain

It is hydrophobic and inserts into lipid bilayers

What structural change drives viral membrane fusion

Formation of a hairpin composed of alpha helices

How does the viral hairpin promote fusion

By pulling the viral and host membranes together

What secondary structure dominates viral fusion proteins

Alpha helices

What is a coiled-coil

A structure formed by multiple alpha helices packing via hydrophobic stripes

How many degrees does each residue advance in an alpha helix

Approximately 100 degrees

Which amino acid positions form hydrophobic stripes in coiled-coils

Positions 1 4 and 7

Why are coiled-coils energetically important

Their high stability releases energy that drives membrane fusion

What experimental system demonstrates fast cellular fusion

Mast cell exocytosis

How is membrane fusion detected electrophysiologically

By measuring increases in membrane capacitance

What does membrane capacitance reflect

The surface area of the membrane

What is the timescale of membrane fusion in cells

Approximately one millisecond

What property of fusion shows it can be reversible

Fusion pores can open and close repeatedly

Who reconstituted intra Golgi transport biochemically

Rothman and colleagues

Why is in vitro reconstitution important

It allows biochemical manipulation and identification of fusion factors

What reagent was used to identify fusion proteins

N ethylmaleimide NEM

How does NEM inhibit fusion

By modifying essential cysteine residues in proteins

What did cytosol addition after NEM treatment show

A cytosolic factor is required for fusion

What protein was identified as NEM sensitive

NSF

What type of protein is NSF

A AAA ATPase and unfoldase

How fast does NSF hydrolyze ATP

Approximately one ATP per second

Why can NSF not directly drive fast fusion

Its ATPase activity is too slow