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 apparatus Plasma ER and Golgi Cisternae

Assembly of a clathrin coat triskelion Coated pits and vesicles on the cytosolic surface of membranes Freeze-etch 36 triskelions 12 pentagons 6 hexagons Inner layer binds adaptins

Adaptin binds to cargo receptor and clathrin triskelion Dynamin pinches off the bud Four types of adaptins Auxillin-activated ATPase is required To remove the clathrin coat Vesicles can have different shapes

Dynamin pinches of the vesicles GTPase Shibire mutant has coated pits but no budding off of synaptic vesicles

ARF proteins: COPI&clathrin Sar1 protein: COPII GTP causes Sar1 to Bind to membrane Assembly and disassembly of coat by GTPases Coat-recruitment GTPases GTPase works like a timer And cause disassembly shortly After the budding is completed

Guidance of vesicular transport SNAREs: specificity and fusion Rab GTPases: initial docking and tethering of vesicles to target membranes and matching of v- and t- SNAREs

SNARE proteins guide vesicular transport 20 SNAREs, v-SNAREs, t-SNAREs

SNAREs specify compartment identity and control specificity 4  helices in trans-SNARE complexes

Rab proteins ensure the specificity of vesicle docking >30 Rabs On 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) Selective process

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 proteins that “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 complex oligosaccharides high-mannose oligosaccharides

Oligosaccharide processing in the ER and the Golgi

Why glycosylation? Folding Transport Stability Recognition Regulatory roles (Notch)

Histochemical stains: biochemical Compartmentalization of the Golgi Functional compartmentalization

Transport through the Golgi may occur by vesicular transport or cisternal maturation (not mutually exclusive) Collagen rods Scales in algae

Summary 1.Vesicular transport, biosynthetic-secretory and endocytic 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.