Dynamic or Stable Interactions of Influenza Hemagglutinin Mutants with Coated Pits

doi:10.1074/jbc.270.36.21075

Dynamic or Stable Interactions of Influenza Hemagglutinin Mutants with Coated Pits

DEPENDENCE ON THE INTERNALIZATION SIGNAL BUT NOT ON AGGREGATION (*)

From the (1)Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel and the
(2)University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235

^§ To whom correspondence should be addressed. Tel.: 972-3-6409053; Fax: 972-3-6407643.

Abstract

Measurements of the lateral mobility of native and mutated membrane proteins, combined with treatments that alter clathrin lattice structure, are capable of characterizing their interactions with coated pits in live cells (Fire, E., Zwart, D. E., Roth, M. G., and Henis, Y. I.(1991) J. Cell Biol. 115, 1585-1594). To explore the dependence of these interactions on the internalization signal and the aggregation state of the protein, we have extended this approach to investigate the interactions between coated pits and several influenza hemagglutinin (HA) mutants, which differ in the internalization signals in their short cytoplasmic tails. The lack of internalization signals in the trimeric wild-type HA enables a direct comparison between specific internalization signals introduced singly in each mutant. We have selected for these studies HA mutants that showed different internalization rates and varied in their tendency to aggregate into complexes larger than trimers. Our results indicate that the mode of interaction with coated pits (transient association-dissociation versus stable entrapment) depends on the internalization signal and affects the internalization efficiency. Mutants that contain a strong internalization signal and undergo fast endocytosis were entrapped in coated pits for the entire duration of the lateral mobility measurement, suggesting stable association with (slow dissociation from) coated pits. A mutant with a suboptimal internalization signal, which was internalized 10-fold slower, exhibited transient interactions with coated pits. Both types of interactions disappeared or were significantly reduced upon disruption of the clathrin lattices under hypertonic conditions, and were modulated following the “freezing” of coated pits by cytosol acidification. Unlike the dependence on the cytoplasmic internalization signal, the interactions with coated pits did not depend on the aggregation state (measured by sucrose gradient centrifugation after solubilization in n-octylglucoside) of the mutants.

Footnotes

↵* This work was supported in part by grants from the German-Israeli Foundation for Scientific Research and Development (to Y. I. H.), the Human Frontier Science Program Organization Grant RG355/93, and National Institutes of Health Grant GM37547 (to M. G. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

EGF

epidermal growth factor

HA

influenza hemagglutinin

TMR

tetramethylrhodamine

STI

soybean trypsin inhibitor

HBSS

Hanks' balanced salt solution

BSA

bovine serum albumin

PAGE

polyacrylamide gel electrophoresis

DMEM

Dulbecco's modified Eagle's medium

FPR

fluorescence photobleaching recovery

wt

wild type

KA

potassium-amiloride.
- Received May 11, 1995.
- Revision received July 6, 1995.