1. Vesicle Formation at the Plasma Membrane and Trans-Golgi Network: The Same but Different M. A. McNiven, et al., Science 313, 1591 (2006) Department of Biochemistry and Molecular Biology and the Miles and Shirley Fiterman Center for Digestive Diseases, Mayo Clinic College of Medicine, Rochester Liangfang Zhao, Yanqing Lin & Zhiling Yao Life Science College, CNU 2007.10.30

2. Abbreviations PM: Plasma Membrane PM: Plasma Membrane TGN: Trans-Golgi Network TGN: Trans-Golgi Network CCV: Clathrin-Coated Vesicle CCV: Clathrin-Coated Vesicle APs: Adaptor Proteins APs: Adaptor Proteins EGF: Epidermal Growth Receptor EGF: Epidermal Growth Receptor GGAs: Golgi-localized, γ- GGAs: Golgi-localized, γ-earcontaining, Adenosine diphosphate Ribosylation Factor (ARF) PIP2: PhosphatidylInositol 4,5-bisPhosphate PI4P: PhosphatidylInositol 4-monoPhosphate UIMs: Ubiquitin Interacting Motifs

3. Overview: yin & yang relationship Sorting, packaging, vesiculation, transport of membrane and protein cargo Sorting, packaging, vesiculation, transport of membrane and protein cargo active exchange of membranes & protein cargo; active exchange of membranes & protein cargo; highly conserved mechanisms in vesicle formation & scission highly conserved mechanisms in vesicle formation & scission In and out of the cell In and out of the cell Membrane continuum Membrane continuum

4. Index I. similarities and variations in sorting mechanisms I. similarities and variations in sorting mechanisms I. similarities and variations in sorting mechanisms I. similarities and variations in sorting mechanisms II. Lipid-membrane binding, bending and pinching II. Lipid-membrane binding, bending and pinching II. Lipid-membrane binding, bending and pinching II. Lipid-membrane binding, bending and pinching III. Vesicle scission III. Vesicle scission III. Vesicle scission III. Vesicle scission

5. I. similarities and variations in sorting mechanisms Similarities in protein-sorting mechanisms at the PM and TGN Similarities in protein-sorting mechanisms at the PM and TGN Similarities in protein-sorting mechanisms at the PM and TGN Similarities in protein-sorting mechanisms at the PM and TGN Variations of a common sorting machine Variations of a common sorting machine Variations of a common sorting machine Variations of a common sorting machine

6. 1.Similarities in protein-sorting mechanisms at the PM and TGN Key functions: selective sequestration and packaging of protein cargo Key functions: selective sequestration and packaging of protein cargo Central scaffold: clathrin and its associated coat proteins Central scaffold: clathrin and its associated coat proteins

7. APs and monomeric clathrin-associated sorting proteins “ first line ” of cargo-sequestration specificity “ first line ” of cargo-sequestration specificity AP1-4 AP1-4 similar organization, subunits, structure; similar organization, subunits, structure; different cellular localization patterns, vesicle-formation processes different cellular localization patterns, vesicle-formation processes

8. AP: mediate protein sorting Recognition of sorting motifs of the cytosolic domains of transmembrane proteins Recognition of sorting motifs of the cytosolic domains of transmembrane proteins Three classes Three classes Asn-Pro-X-Tyr: AP-2 at the PM while interact with non-AP-2 Asn-Pro-X-Tyr: AP-2 at the PM while interact with non-AP-2 Tyr-X-X-Φ: μsubunit of all 4 APs Tyr-X-X-Φ: μsubunit of all 4 APs [Asp-Glu] X-X-X-Leu [Leu-Ile]: AP1-3 yet different preferences [Asp-Glu] X-X-X-Leu [Leu-Ile]: AP1-3 yet different preferences

9. Conclusion for PART 1 Different affinities between leaving and being internalized Different affinities between leaving and being internalized

10. 2.Variations of a common sorting machine Several layers of regulation and targeting: provide specificity Several layers of regulation and targeting: provide specificity Different lipids & phosphoinositides; small GTPases; GGAs Different lipids & phosphoinositides; small GTPases; GGAsGGAs

11. Lipids Cholesterol, sphingoipids, phosphoinositides Cholesterol, sphingoipids, phosphoinositides Rafts (lipid microdomains) Rafts (lipid microdomains) mediate clathrin-independent vesicle formation mediate clathrin-independent vesicle formation participate in sorting at the TGN participate in sorting at the TGN ratio differ: maintain the participation of membrane microdomains ratio differ: maintain the participation of membrane microdomains Form vesicle: inositol phospholipids Form vesicle: inositol phospholipids different combinations of phospho-modifications: PIP 2 & PI4P(LESS TIDY) different combinations of phospho-modifications: PIP 2 & PI4P(LESS TIDY)

12. Same phosphoinositide: essential in multiple distinct vesicle formation processes at both sides

13. Small GTPases Regulate secretory & endocytic vesicle formation Regulate secretory & endocytic vesicle formation TGN targeting of AP-1: Active ARF1 & Golgi-enriched PI4P TGN targeting of AP-1: Active ARF1 & Golgi-enriched PI4P PM CCV formation: ARF family dependent in some cases PM CCV formation: ARF family dependent in some cases

14. GGAs: function along with the AP- based sorting system Recruitment to Golgi: ARF dependent Recruitment to Golgi: ARF dependent Generate CCVs ’ subset which targets to lysosomes: function along with AP-1 Generate CCVs ’ subset which targets to lysosomes: function along with AP-1 Mediate sorting of lysosome-targeted proteins: bind to different dileucine motifs Mediate sorting of lysosome-targeted proteins: bind to different dileucine motifs AP-1: [DE]XXXL[LI] AP-1: [DE]XXXL[LI] GGAs: Asp-X-X-Leu-Leu GGAs: Asp-X-X-Leu-Leu

15. Ubiquitin modification TGN: Initiate nascent proteins sorting TGN: Initiate nascent proteins sorting PM: Sort endocytosed proteins to endosomes & lysosomes PM: Sort endocytosed proteins to endosomes & lysosomes At both vesicle-formation sites: At both vesicle-formation sites: PM: Eps15 & Epsin----sequester & sort activated EGF receptors PM: Eps15 & Epsin----sequester & sort activated EGF receptors bind each other & AP-2 bind each other & AP-2 contain UIMs contain UIMsUIMs TGN: GGAs & Eps15 TGN: GGAs & Eps15 Eps15: AP-1 Eps15: AP-1

16. costain