Vesicle Formation at the Vesicle Formation at the Plasma Membrane and Trans-Plasma Membrane and Trans-Golgi Network: The Same but Golgi Network: The Same but

DifferentDifferent

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

AbbreviationsAbbreviations

• PM: Plasma MembranePM: Plasma Membrane• TGN: Trans-Golgi NetworkTGN: Trans-Golgi Network• CCV: Clathrin-Coated VesicleCCV: Clathrin-Coated Vesicle• APs: Adaptor ProteinsAPs: Adaptor Proteins• EGF: Epidermal Growth ReceptorEGF: Epidermal Growth Receptor• GGAsGGAs: Golgi-localized, : Golgi-localized, γγ --earcontaining,

Adenosine diphosphate Ribosylation Factor (ARF)

•PIP2: PhosphatidylInositol 4,5-bisPhosphate•PI4P: PhosphatidylInositol 4-

monoPhosphate •UIMs: Ubiquitin Interacting Motifs

Overview: yin & yang Overview: yin & yang relationshiprelationship

• Sorting, packaging, vesiculation, transporSorting, packaging, vesiculation, transport of membrane and protein cargot of membrane and protein cargo

active exchange of membranes & protein active exchange of membranes & protein cargo;cargo;

highly conserved mechanisms in vesicle highly conserved mechanisms in vesicle formation & scissionformation & scission

• In and out of the cellIn and out of the cell• Membrane continuumMembrane continuum

IndexIndex

• I. similarities and variations in sortinI. similarities and variations in sorting mechanismsg mechanisms

• II. Lipid-membrane binding, bending II. Lipid-membrane binding, bending and pinchingand pinching

• III. Vesicle scissionIII. Vesicle scission

I. similarities and variations in I. similarities and variations in sorting mechanismssorting mechanisms

• Similarities in protein-sorting mSimilarities in protein-sorting mechanisms at the PM and TGNechanisms at the PM and TGN

• Variations of a common sorting Variations of a common sorting machinemachine

1.Similarities in protein-1.Similarities in protein-sorting mechanisms at the sorting mechanisms at the PM and TGNPM and TGN

•Key functions: selective Key functions: selective sequestration and packaging of sequestration and packaging of protein cargoprotein cargo

•Central scaffold: clathrin and its Central scaffold: clathrin and its associated coat proteinsassociated coat proteins

APs and monomeric clathrin-APs and monomeric clathrin-associated sorting proteinsassociated sorting proteins

• ““first line” of cargo-sequestration specificityfirst line” of cargo-sequestration specificity

• AP1-4AP1-4

similar organization, subunits, structure;similar organization, subunits, structure;

different cellular localization patterns, different cellular localization patterns, vesicle-formation processesvesicle-formation processes

AP: mediate protein AP: mediate protein sortingsorting

• Recognition of sorting motifs of the Recognition of sorting motifs of the cytosolic domains of transmembrane cytosolic domains of transmembrane proteinsproteins

• Three classesThree classes

Asn-Pro-X-Tyr: AP-2 at the PM while Asn-Pro-X-Tyr: AP-2 at the PM while interact with non-AP-2 interact with non-AP-2

Tyr-X-X-Tyr-X-X-ΦΦ: : μμsubunit of all 4 APssubunit of all 4 APs

[Asp-Glu] X-X-X-Leu [Leu-Ile]: AP1-3 yet [Asp-Glu] X-X-X-Leu [Leu-Ile]: AP1-3 yet different preferencesdifferent preferences

Conclusion for PART 1Conclusion for PART 1

Different affinities Different affinities between leaving and between leaving and

being internalizedbeing internalized

2.Variations of a common 2.Variations of a common sorting sorting machinemachine

• Several layers of regulation and Several layers of regulation and targeting: provide specificitytargeting: provide specificity

• Different lipids & phosphoinositides; Different lipids & phosphoinositides; small GTPases; small GTPases; GGAsGGAs

LipidsLipids• Cholesterol, sphingoipids, Cholesterol, sphingoipids,

phosphoinositidesphosphoinositides• Rafts (lipid microdomains)Rafts (lipid microdomains) mediate clathrin-independent vesicle mediate clathrin-independent vesicle

formation formation participate in sorting at the TGN participate in sorting at the TGN ratio differratio differ: maintain the participation : maintain the participation

of membrane microdomainsof membrane microdomains• Form vesicle: inositol phospholipidsForm vesicle: inositol phospholipids

different combinationsdifferent combinations of of phospho-modifications: phospho-modifications:

PIPPIP22 & & PI4P(LESS TIDY) PI4P(LESS TIDY)

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

Small GTPasesSmall GTPases

•Regulate secretory & endocytic Regulate secretory & endocytic vesicle formationvesicle formation

•TGN targeting of AP-1: Active TGN targeting of AP-1: Active ARF1 & Golgi-enriched PI4PARF1 & Golgi-enriched PI4P

•PM CCV formation: ARF family PM CCV formation: ARF family dependent in some casesdependent in some cases

GGAs: function along with the GGAs: function along with the AP-based sorting systemAP-based sorting system

• Recruitment to Golgi: ARF dependentRecruitment to Golgi: ARF dependent

• Generate CCVs’ subset which targets to Generate CCVs’ subset which targets to lysosomes: function along with AP-1lysosomes: function along with AP-1

• Mediate sorting of lysosome-targeted Mediate sorting of lysosome-targeted proteins: bind to proteins: bind to differentdifferent dileucine dileucine motifsmotifs

AP-1: [DE]XXXL[LI]AP-1: [DE]XXXL[LI]

GGAs: Asp-X-X-Leu-LeuGGAs: Asp-X-X-Leu-Leu

Ubiquitin Ubiquitin modificationmodification

• TGN: Initiate nascent proteins sorting TGN: Initiate nascent proteins sorting

• PM: Sort endocytosed proteins to endosomes PM: Sort endocytosed proteins to endosomes & lysosomes& lysosomes

• At both vesicle-formation sites:At both vesicle-formation sites:

PM: Eps15 & Epsin----sequester & sort PM: Eps15 & Epsin----sequester & sort activated EGF receptorsactivated EGF receptors

bind each other & AP-2bind each other & AP-2

contain contain UIMsUIMs

TGN: GGAs & Eps15TGN: GGAs & Eps15

Eps15: AP-1Eps15: AP-1

costain