Clathrin assembly protein AP-2 induces aggregation of membrane vesicles: A possible role for AP-2 in endosome formation

Kenneth A Beck, Michael Chang, Frances M. Brodsky, James H. Keen

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

We have examined the in vitro behavior of clathrin-coated vesicles that have been stripped of their surface coats such that the majority of the clathrin is removed but substantial amounts of clathrin assembly proteins (AP) remain membrane-associated. Aggregation of these stripped coated vesicles (s-CV) is observed when they are placed under conditions that approximate the pH and ionic strength of the cell interior (pH 7.2, ∼100 mM salt). This s-CV aggregation reaction is rapid (t1/2 ≤ 0.5 min), independent of temperature within a range of 4-37°C, and unaffected by ATP, guanosine-5′-Ο-(3-thiophosphate), and in particular EGTA, distinguishing it from Ca2+-dependent membrane aggregation reactions. The process is driven by the action of membrane-associated AP molecules since partial proteolysis results in a full loss of activity and since aggregation is abolished by pretreatment of the s-CVs with a monoclonal antibody that reacts with the α subunit of AP-2. However, vesicle aggregation is not inhibited by PPPi, indicating that the previously characterized polyphosphate-sensitive AP-2 self-association is not responsible for the reaction. The vesicle aggregation reaction can be reconstituted: liposomes of phospholipid composition approximating that found on the cytoplasmic surfaces of the plasma membrane and of coated vesicles (70% L-α-phosphatidylethanolamine (type I-A), 15% L-α-phosphatidyl-L-serine, and 15% L-α-phosphatidylinositol) aggregated after addition of AP-2, but not of AP-1, AP-3 (AP180), or pure clathrin triskelions. Aggregation of liposomes is abolished by limited proteolysis of AP-2 with trypsin. In addition, a highly purified AP-2a preparation devoid of β causes liposome aggregation, whereas pure β subunit does not, consistent with results obtained in the s-CV assay which also indicate the involvement of the α subunit. Using a fluorescence energy transfer assay we show that AP-2 does not cause fusion of liposomes under physiological solution conditions. However, since the fusion of membranes necessarily requires the close opposition of the two participating bilayers, the AP-2-dependent vesicle aggregation events that we have identified may represent an initial step in the formation and fusion of endosomes that occur subsequent to endocytosis and clathrin uncoating in vivo.

Original languageEnglish (US)
Pages (from-to)787-796
Number of pages10
JournalJournal of Cell Biology
Volume119
Issue number4
StatePublished - Nov 1992
Externally publishedYes

ASJC Scopus subject areas

  • Cell Biology

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