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© The Rockefeller University Press, 0021-9525/2002/4/315 $5.00
The Journal of Cell Biology, Volume 157, Number 2, April 15, 2002 315-326

Sla1p serves as the targeting signal recognition factor for NPFX_(1,2)D-mediated endocytosis

Department of Biological Chemistry, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095

Address correspondence to Gregory S. Payne, Dept. of Biological Chemistry, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095-1737. Tel.: (310) 206-3121. Fax: (310) 206-5272. E-mail: gpayne{at}mednet.ucla.edu

Efficient endocytosis requires cytoplasmic domain targeting signals that specify incorporation of cargo into endocytic vesicles. Adaptor proteins play a central role in cargo collection by linking targeting signals to the endocytic machinery. We have characterized NPFX_(1,2) (NPFX_[1,2]D) targeting signals and identified the actin-associated protein Sla1p as the adaptor for NPFX_(1,2)D-mediated endocytosis in Saccharomyces cerevisiae. 11 amino acids encompassing an NPFX_(1,2)D sequence were sufficient to direct uptake of a truncated form of the pheromone receptor Ste2p. In this context, endocytic targeting activity was not sustained by conservative substitutions of the phenylalanine or aspartate. An NPFX_1,2D-related sequence was identified in native Ste2p that functions redundantly with ubiquitin-based endocytic signals. A two-hybrid interaction screen for NPFX_(1,2)D-interacting proteins yielded SLA1, but no genes encoding Eps15 homology (EH) domains, protein modules known to recognize NPF peptides. Furthermore, EH domains did not recognize an NPFX_(1,2)D signal when directly tested by two-hybrid analysis. SLA1 disruption severely inhibited NPFX_(1,2)D-mediated endocytosis, but only marginally affected ubiquitin-directed uptake. NPFX_(1,2)D-dependent internalization required a conserved domain of Sla1p, SLA1 homology domain, which selectively bound an NPFX_(1,2)D-containing fusion protein in vitro. Thus, through a novel NPF-binding domain, Sla1p serves as an endocytic targeting signal adaptor, providing a means to couple cargo with clathrin- and actin-based endocytic machineries.

Key Words: endocytosis; SLA1; NPF; adaptor; clathrin

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