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© The Rockefeller University Press, 0021-9525/1999//457 $5.00
The Journal of Cell Biology, Volume 145, Number 3, , 1999 457-468

Regular Articles

Trafficking of an Acylated Cytosolic Protein: Newly Synthesized p56lck Travels to the Plasma Membrane via the Exocytic Pathway



Marie-José J.E. Bijlmakers and Mark Marsh

MRC Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, London WC1E 6BT, United Kingdom

The Src-related tyrosine kinase p56lck (Lck) is primarily expressed in T lymphocytes where it localizes to the cytosolic side of the plasma membrane and associates with the T cell coreceptors CD4 and CD8. As a model for acylated proteins, we studied how this localization of Lck is achieved. We followed newly synthesized Lck by pulse–chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30–45 min later. Membrane-binding kinetics are similar in CD4/CD8-positive and CD4/CD8-negative cells. In CD4-positive T cells, the interaction with CD4 rapidly follows membrane association of Lck. Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A. These data indicate that the initial association of newly synthesized Lck with CD4, and therefore with membranes, occurs on intracellular membranes of the exocytic pathway. From this location Lck is transported to the plasma membrane.

Key Words: p56lck • acylation • targeting • CD4 • protein sorting

Abbreviations used in this paper: BFA, Brefeldin A; Endo H, endoglycosidase H; Lck, p56lck; TcR, T cell receptor; wt, wild-type.

We especially thank Jannie Borst for providing the anti-Lck serum which was crucial for immunoprecipitations, Karin Römisch and Kate Bowers for critically reading the manuscript, and our colleagues in the MRC-LMCB for helpful discussions. We would further like to thank Philippe Benaroch (Curie Institut, Paris, France) for Jurkat cells.

The work was supported by the UK Medical Research Council and by a long-term EMBO fellowship to M.-J.J.E. Bijlmakers.


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