Summary

1. Rough ER and smooth ER;2. Signal hypothesis, translocation into ER;3. Single-span and multi-span membrane proteins;4. Glycosylation;5. Protein folding;6. Lipid synthesis

Lecture 8 Vesicular trafficking

from ER to Golgi

Endocytic and biosynthetic-secretory pathways

Transport vesicles

(Ten or more chemically distinct, membrane-enclosed compartments)

The biosynthetic-secretory and endocytic pathways

Various types of coated vesicles

Golgi apparatusPlasma

ER and Golgi Cisternae

Assembly of a clathrin coat

triskelionCoated pitsand vesicleson the cytosolic surface of membranes

Freeze-etch

36 triskelions12 pentagons6 hexagons

Inner layer binds adaptins

Adaptin binds to cargo receptor and clathrin triskelion

Dynamin pinches off the bud

Four types of adaptinsAuxillin-activatedATPase is requiredTo remove the clathrin coat

Vesicles can have different shapes

Dynamin pinches of the vesicles

GTPase

Shibire mutanthas coated pitsbut no budding offof synaptic vesicles

ARF proteins: COPI&clathrinSar1 protein: COPII

GTP causes Sar1 toBind to membrane

Assembly and disassembly of coat by GTPases

Coat-recruitment GTPases GTPase works like a timerAnd cause disassembly shortlyAfter the budding is completed

Guidance of vesicular transport

SNAREs: specificity and fusion

Rab GTPases: initial docking and tethering of vesiclesto target membranes and matching of v- and t- SNAREs

SNARE proteins guide vesicular transport20 SNAREs, v-SNAREs, t-SNAREs

SNAREs specify compartment identity and control specificity

4 helicesin trans-SNAREcomplexes

Rab proteins ensure the specificity of vesicle docking

>30 RabsOn cytosolic surface

C-terminal regions are variable:Bind to other proteins, including GEFs

SNAREs may mediate membrane fusion

SNARE complex

After docking

The entry of enveloped viruses into cells

HIV

Similar to SNAREs

Proteins leave the ER in COPII-coated transport vesicles

ER exit sites(no ribosomes)

Selectiveprocess

Only properly folded and assembled proteins can leave the ER

Chaperones cover up exit signals

Homotypic membrane fusion

to form vesicular tubular clusters

Vesicular tubular clusters

Lacks many of the ER proteins

COPI-coated

Retrograde transport:

Short-lived

carry back the ER resident proteinsthat “leaked” out

ER retrieval signals: KKXX in ER membrane proteins,

KDEL sequence in soluble ER resident proteins

Membrane proteins in Golgi and ER have shorter TM domains (15 aa)Cholesterol

pH controls affinity of KDEL receptors

Ordered series of Golgi compartments

Cisternae, tubular connections

Plant cell

MT is required to localize near the cell nucleus close to the centrosome (in animal cells)

Plant cells

Two main classes of N-linked glycosilation

core

complexoligosaccharides

high-mannoseoligosaccharides

Oligosaccharide processing in the ER and the Golgi

Why glycosylation?

FoldingTransportStability RecognitionRegulatory roles(Notch)

Histochemical stains: biochemicalCompartmentalization of the Golgi

Functional compartmentalization

Transport through the Golgi may occur by vesiculartransport or cisternal maturation (not mutually exclusive)

Collagen rodsScales in algae

Summary

1. Vesicular transport, biosynthetic-secretory andendocytic pathways;

2. Coated vesicles;3. Coat assembly and disassembly, budding, dynamin,

coat-recruitment GTPases;4. Targeting and fusion by Rab GTPases, SNAREs;5. ER to Golgi: COPII, folding, fusion (cluster), retrograde;6. Golgi apparatus structure and polarity;7. Continuation of glycosylation;8. Compartmentalization of Golgi cisternae;9. By now we have introduced gated transport, transmembrane

transport and vesicular transport.