Chapter 13: Vesicular Transport Part 1

Transport vesicle

• Contains membrane components and soluble lumenal molecules (called cargo)

• Transport vesicles bud from one compartment and fuse to another; selective process

3 pathways of vescular transport

• secretory pathway

• endocytic pathway

• retrieval pathway

Secretory Pathway

•  (red arrows)

• leads outward from the ER to the Golgi apparatus and cell surface, with a side route leading to lysosomes 

Endocytic Pathway

•  (green arrows)

• leads inward from plasma membrane

Retrieval Pathways

• blue arrows

• balance flow of membrane between compartments (opposite direction)

Golgi Apparatus

• processing center of the cell:

• responsible for Transporting, modifying, and packaging proteins and lipids into vesicles for delivery to targeted destinations

Structure of Golgi apparatus

•  Consists of a collection of flattened, membrane-enclosed compartments called cisternae (stack of pancakes), each golgi stack typically consists of 4-6 cisternae

  • tubular connections between corresponding cisternae link stacks. This forms a single complex (near cell nucleus)

• Cargo enters through the cis face and leaves through the trans face in secretory vesicles

Two layers of a vesicle coat

• Inner coat layer concentrates specific membrane proteins in patch, selects membrane molecules for transport

  • What plays a role in packaging content

• Outer coat resembles a basket-like lattice, deforms the membrane patch and shapes the vesicle

What would happen if vesicles had no outer clathrin coat

• Vesicles cannot form properly without a clathrin cage

• endocytosis will be inhibited

Clathrin coated (endocytic) vesicles

• from PM and between endosomal and Golgi compartments

• Clathrin triskelions assemble into basketlike framework (ie hexagons) to form coated pits (buds) on cytosolic surface of membranes

• Plasma membrane → endosomes and trans-Golgi network → endosomes

Adaptor proteins

form a discrete inner layer of the coat (bridge between clathrin cage and membrane)

bind outer coat to membrane ans trap cargo receptors (select for specific set of proteins)  and package the4m into vesicles

Dynamin

• soluble cytoplasmic protein that assemble at the neck of each bud, as a clathrin coated bud grows

Non-clathrin coat

• observed on isolated Golgi membranes and vesicles in transport assays

COPI coated (Coat proteins)-from Golgi to ER (or backwards)

• Bring back ER resident proteins that have escaped to the golgi

• coat is recruited by ARF1

COPII coated (Coat proteins)- bud from ER (secretory) ; er to golgi

• Mediate transport ro the ER to the cis golgi network

• Proteins leave the ER in COP II vesicles

• SAR1 recruits coat

How dos vesicle budding work

1.Recruitment -A small GTPase is activated and recruits coat components to the membrane. Examples: Sar1 for COPII and ARF1 for COPI.

2. Cargo selection -  Cargo proteins or cargo receptors are recognized by adaptor or coat proteins.

3 Membrane bending - Coat proteins assemble into a curved structure, concentrating cargo and shaping the membrane into a bud.

4. Scission and uncoating - The bud separates from the membrane. Dynamin drives scission at many plasma-membrane clathrin buds; other vesicles use different mechanisms. The coat is subsequently removed so the vesicle can interact with its target.

Exit signals

• Molecular features that allow proteins to be selected for transport out of the ER; help the cell recognize proteins that are ready to move to the cis-golgi 

Do resident ER proteins have exit signals

• No? Generally no

  • Instead they can ente the normal export pathway and tnen be retrieved back to the ER

  • Two mechanisms:
    Retrieval - some er resident proteins accidentally leave the ER 

  • Retention - other proteins have features that keep them in the er, like membrane anchors or retention signals

Rab

 involved in direction

SNARE Proteins

mediate membrane fusion

Rab

• GTPases

• Associated with one or more membrane-enclosed organelles

• Selective distribution across t membranes (ideal marker)

GDP- Rab

 (inactive and bound to another protein: Rab-GDP dissociatio inhibitor, GDI, GDI), protein soluble

GTP-Rab

(active and associated with membrane of organelle or transport vesicle)

Snares

• Mediate membrane fusion

• V and T snares interact to form

• Trans SNARE complex

• Helices overcome hydration shell

• NFS protein (ATPase) separates snares

• after fusion

Membrane fusion

• For vesicle transport to operate normally, vesicles must incorporate appropriate SNARE and Rab proteins

• Ruptture of the new bilayer completes fusion 

• SNARE must be pried apart before it can function again -> done by NSF: N-ethylmaleimide-sensitive fusion protein (ATPase).

ER to Golgi Transport

Homotypic fusion

• When two membranes from the same type of organelle fuse together, combining  contents and membrane

Retrieval pathways

• As soon as vesicular tubular clusters form, they bud off transport vesicles of their own, which are COPI coated vesicles

• They function as a retrieval pathway (bring back resident proteins, SNAREs, cargo receptors)

KDEL

• cycles between golgi and er

• Affinity for KDEL sequence must differ in each compartment

Where should KDEL e high v low

• High affinity in the Golgi: The KDEL receptor binds tightly to ER-resident proteins carrying the KDEL retrieval signal, allowing them to be captured for return to the ER.

• Low affinity in the ER: The receptor releases the KDEL-containing protein once it returns to the ER.

Why? The difference in conditions between the Golgi and ER—especially pH—helps the receptor bind cargo in the Golgi and release it in the ER.

Memory trick: Golgi = grab KDEL proteins; ER = eject them.

Oligosaccharide Processing in Golgi

Proteins entering from the ER undergo ordered oligosaccharide modification as they pass through the cis, medial, and trans Golgi cisternae. Each cisterna contains characteristic membrane-bound processing enzymes. 

Why are proteins glycosylated?

1. To promote protein folding 

2. More resistance to digestion by proteases

3. Cell adhesion (selectins) in leukocyte rolling

4. Immune function: presence of sugar can modify antigenic properties

Cisternal Maturation Model of Transport in the Golgi Apparatus

each cisterna matures over time, acquiring new components (as well as red fluorescence) as it matures, and recycling old components (green fluorescence) to the less mature cisternae by retrograde vesicle transpo

Vesicle transport Model

predicts that each cisterna remains stationary and fluoresces either red (or yellow) or green during the entire period