Rapid Plasma Membrane Anchoring of Newly Synthesized p59 fyn: Selective Requirement for NH2-Terminal Myristoylation and Palmitoylation at Cysteine-3

doi:10.1083/jcb.136.5.1023

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© The Rockefeller University Press, 0021-9525/1997//1023 $5.00
The Journal of Cell Biology, Volume 136, Number 5, , 1997 1023-1035

Article

Rapid Plasma Membrane Anchoring of Newly Synthesized p59 ^fyn: Selective Requirement for NH₂-Terminal Myristoylation and Palmitoylation at Cysteine-3

Wouter van 't Hof and Marilyn D. Resh

Cell Biology and Genetics Program, Sloan-Kettering Institute for Cancer Research, New York, 10021

The trafficking of Src family proteins after biosynthesis ispoorly defined. Here we studied the role of dual fatty acylationwith myristate and palmitate in biosynthetic transport of p59^fyn.Metabolic labeling of transfected COS or NIH 3T3 cells with[³⁵S]methionine followed by analysis of cytosolic and totalmembrane fractions showed that Fyn became membrane bound within5 min after biosynthesis. Newly synthesized Src, however, accumulatedin the membranes between 20– 60 min. Northern blottingdetected Fyn mRNA specifically in soluble polyribosomes andsoluble Fyn protein was only detected shortly (1–2 min)after radiolabeling. Use of chimeric Fyn and Src constructsshowed that rapid membrane targeting was mediated by the myristoylatedNH₂-terminal sequence of Fyn and that a cysteine at position3, but not 6, was essential. Examination of G ${alpha}$ _o-, G ${alpha}$ _s-, or GAP43-Fynfusion constructs indicated that rapid membrane anchoring isexclusively conferred by the combination of N-myristoylationplus palmitoylation of cysteine-3. Density gradient analysis colocalized newly synthesized Fyn with plasma membranes. Interestingly,a 10–20-min lag phase was observed between plasma membranebinding and the acquisition of non-ionic detergent insolubility.We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylationand plasma membrane anchoring, and a slower partitioning intodetergent-insoluble membrane subdomains. These results serveto define a novel trafficking pathway for Src family proteinsthat are regulated by dual fatty acylation.

Abbreviations used in this paper: IC-13, 13-[¹²⁵I] iodotridecanoic acid; IC-16, 16-[¹²⁵I] iodohexadecanoic acid; NMT, N-myristoyl acyltransferase; PAT, palmitoyl acyltransferase; SH, Src Homology domain; TBR-I, transforming growth factor β type-I receptor.

Please address all correspondence to M.D. Resh, Cell Biologyand Genetics Program, Sloan-Kettering Institute for Cancer Research,1275 York Avenue, Box 143, New York, NY 10021. Tel.: (212)639-2514. Fax: (212) 717-3317. E-Mail: m-resh{at}ski.mskcc.org

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