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

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.