Coupled Inositide Phosphorylation and Phospholipase D Activation Initiates Clathrin-coat Assembly on Lysosomes

doi:10.1074/jbc.274.25.17794

Coupled Inositide Phosphorylation and Phospholipase D Activation Initiates Clathrin-coat Assembly on Lysosomes*

From the ^‡Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 and the ^§Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706

Abstract

Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, we show that adaptor recruitment and coat initiation requires phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) synthesis. PtdIns(4,5)P₂ is generated on lysosomes by the sequential action of a lysosome-associated type II phosphatidylinositol 4-kinase and a soluble type I phosphatidylinositol 4-phosphate 5-kinase. Phosphatidic acid, which potently stimulates type I phosphatidylinositol 4-phosphate 5-kinase activity, is generated on the bilayer by a phospholipase D1-like enzyme located on the lysosomal surface. Quenching phosphatidic acid function with primary alcohols prevents the synthesis of PtdIns(4,5)P₂ and blocks coat assembly. Generating phosphatidic acid directly on lysosomes with exogenous bacterial phospholipase D in the absence of ATP still drives adaptor recruitment and limited coat assembly, indicating that PtdIns(4,5)P₂functions, at least in part, to activate the PtdIns(4,5)P₂-dependent phospholipase D1. These results provide the first direct evidence for the involvement of anionic phospholipids in clathrin-coat assembly on membranes and define the enzymes responsible for the production of these important lipid mediators.

Footnotes

↵* This work was supported by National Institutes of Health post-doctoral training Grant T32 HL07088 (to L. S. A.) and Grant R01 DK53249 (to L. M. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to Monette Springer for continual encouragement, kindness, and generosity.
↵¶ To whom correspondence should be addressed: Div. of Hematology, Box 8125, Dept. of Internal Medicine, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-747-3711; Fax: 314-362-8813; E-mail: ltraub{at}im.wustl.edu.
↵2 L. M. Traub, unpublished results.
↵3 L. S. Arneson and L. M. Traub, unpublished results.
Abbreviations:

TGN

trans-Golgi network

AP

adaptor protein

ARF

ADP-ribosylation factor

GroPIns

glycerophosphorylinositol

GST

glutathione S-transferase

GTPγS

guanosine 5′-O-(3-thio)triphosphate

HC

heavy chain

LC

light chain

MPR

mannose 6-phosphate receptor

PI4K

phosphatidylinositol 4-kinase

PIP5K

phosphatidylinositol 4-phosphate 5-kinase

PKCα

protein kinase Cα

PLD

phospholipase D

PtdBut

phosphatidylbutanol

PtdCho

phosphatidylcholine

PtdIns

phosphatidylinositol

PtdInsP

phosphatidylinositol phosphate

PtdIns(3)P

phosphatidylinositol 3-phosphate

PtdIns(4)P

phosphatidylinositol 4-phosphate

PtdIns(4,5)P₂

phosphatidylinositol 4,5-bisphosphate

PtdOH

phosphatidic acid

SH3

Src homology domain 3

ATPγS

adenosine 5′-(γ-thio)triphosphate

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis

lgp

lysosomal glycoprotein

HPLC

high performance liquid chromatography

AMP-PNP

adenylyl imidodiphosphate
- Received December 22, 1998.
- Revision received March 11, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.