A Requirement for Ankyrin Binding to Clathrin during Coated Pit Budding

doi:10.1074/jbc.274.50.35908

A Requirement for Ankyrin Binding to Clathrin during Coated Pit Budding*

From the ^‡Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235 and the ^¶Howard Hughes Medical Institute, Department of Cell Biology, Duke University, Durham, North Carolina 27710

Abstract

Recent studies suggest that the mobility of clathrin-coated pits at the cell surface are restricted by an actin cytoskeleton and that there is an obligate reduction in the amount of spectrin on membranes during coated pit budding. The spectrin-actin cytoskeleton associates with membranes primarily through ankyrins, which interact with the cytoplasmic region of numerous integral membrane proteins. We now report that the fourth repeat domain (D4) of ankyrin_R binds to the N-terminal domain of clathrin heavy chain with high affinity. Addition of peptides containing the D4 region inhibited clathrin-coated pit budding in vitro. In addition, microinjection of D4 containing peptides blocked the endocytosis of fluorescent low density lipoprotein (LDL). Ankyrin_R peptides that contained repeat domains other than D4 had no effect on either in vitro budding or internalization of LDL. Finally, immunofluorescence shows that ankyrin is uniformly associated with endosomes that contain fluorescent LDL. These results suggest that ankyrin plays a role in the budding of clathrin-coated pits during endocytosis.

Footnotes

↵* This work was supported by National Institutes of Health Grants DK29808 and HL20948 and the Perot Family Foundation.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.
↵§ To whom correspondence should be addressed: Dept. of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75235-9039. Tel.: 214-648-2576; Fax: 214-648-7577; E-mail: Michaely@utsw.swmed.edu.
Abbreviations:

mAb

monoclonal antibody

LDL

low density lipoprotein

DTT

dithiothreitol

BSA

bovine serum albumin

RIA

radioimmune assay

PBS

phosphate-buffered saline

FITC

fluorescein isothiocyanate

PMCA

3-pyrenemethyl-23,24-dinor-5cholen-22-oate-3β-yl oleate

ALLN

N-Ac-Leu-Leu-norleucinal

HLPPS

human lipoprotein poor serum
- Received June 22, 1999.
- Revision received September 9, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.