The ER and Golgi Modify & Sort Proteins 1

Glycosylation Affects Protein & Lipid Function Serine (S) / Threonine (T) Asparagine (N) Folding Stability Recognition Virtually all cells in body carry glycosylated proteins or lipids. Virtually all secreted or membrane proteins contain sugar modifications. Functions: Protein folding & stability Blood type determinants – A, B, O blood groups – glycosylation of glycophorin Microbes & other pathogens attach and invade Cell-cell adhesion – white blood cells adhere to endothelial cells Bind growth factors – signaling involved in development & disease Regulates protein sorting to different compartments 2

Glycosylation Begins in the ER OST 3

Glycosylation Continues in the Golgi Unique enzymes carry out each step of glycosylation! 4

Two Models for Progression Through Golgi (ERGIC) (ERGIC) Cisternal maturation: COPI vesicles would contain glycosyltransferases that carry out http://publications.nigms.nih.gov/insidethecell/extras/golgi/itc_cisternaematuration.html http://publications.nigms.nih.gov/insidethecell/extras/golgi/itc_vesicularshuttle.html 5

Model 24: COPII Coat Protein Assembly in Vesicular Transport COPII Coat Formation GEF GTP Sec13/31 coat proteins GEF GTP Sar1 GTPase GTP Sec23/24 coat proteins GEF GDP Sar1 GEF GTP GTP GEF Transmembrane & soluble cargo recruitment Cytoplasm GEF ER lumen Define site of vesicle budding Recruit coat proteins Recruit specific vesicle cargo to the budding site Deform the membrane Pinch off to form a vesicle 6

Model 23: Vesicular Transport Through the Secretory Pathway Rough ER COPI Coat (Anterograde) COPII Coat (Anterograde) Golgi Complex cis medial trans COPI Coat (Retrograde) KDEL/KKXX proteins ERGIC Endocytosis Dynein MTOC Kinesin Kinesin Unknown Coat? Exocytosis Dynein 7

Retrieval of ER-Resident Proteins [H+] ER Retention/Retrieval Signals KDEL: Lys-Asp-Glu-Leu Soluble ER proteins KKXX: Lys-Lys-aa-aa Transmembrane ER proteins KDEL ER-resident protein K X KDEL Receptor Golgi Cytoplasm ER retrieval signal is KDEL – constitutive retrieval KDEL binds KDEL Receptor – conditional retrieval KDELR has KKXX at C-terminus that can interact with COPI coat proteins. This signal is hidden until the receptor interacts with a KDEL protein, which causes a conformational change that exposes the KKXX motif. KDELR interacts with KDEL at lower pH in the Golgi. When transported back to the ER, the pH increases and the ligand and receptor dissociate. COPI coat proteins 8

Clathrin Coats Help Vesicles Bud Off from Golgi 9

Intracellular Transport Yildiz, Tomishige, Vale & Selvn. Science (2004) 10

Kinesin, Dynein & Myosin are Molecular Motors Often (not always) (+) end directed Moves along MTs Requires ATP hydrolysis >45 different varieties Dynein (-) end directed Only 1 cytoplasmic dynein heavy chain Myosin (not shown) Moves along actin 17 classes, lots of members 11

Secretory Vesicles Fuse with the Plasma Membrane During Exocytosis 12

Clathrin-Mediated Endocytosis Clathrin (triskelion) – the coat Adapter proteins – recruit cargo & clathrin coat Dynamin (a GTPase) – pinches vesicle off membrane Adapter protein 13