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PMID: 1661731 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits.

The Journal of cell biology ·Vol. 115 ·No. 6 ·1991-12-00 ·Pages 1585-94

Fire E, Zwart DE, Roth MG, Henis YI

Abstract

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.

MeSH Terms
Biological Transport Cell Line Coated Pits, Cell-Membrane/metabolism,ultrastructure Fluorescent Antibody Technique Hemagglutinin Glycoproteins, Influenza Virus Hemagglutinins, Viral/genetics,metabolism Kinetics Mutation Simian virus 40/genetics Temperature Tyrosine/metabolism
Chemicals
Hemagglutinin Glycoproteins, Influenza Virus Hemagglutinins, Viral Tyrosine
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Fire E
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel.
Zwart D E
Roth M G
Henis Y I
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.115.6.1585
Published
1991-12-00
Pages
1585-94
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2289209
Subset
IM
Grants
NIGMS NIH HHS · GM37547 · United States
External Links
PubMed source DOI: 10.1083/jcb.115.6.1585 PMC Full Text View journal details
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