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 NPFX1,2D-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.
Mesh Terms:
Amino Acid Motifs, Amino Acid Sequence, Binding Sites, Carrier Proteins, Conserved Sequence, Cytoskeletal Proteins, Endocytosis, Fungal Proteins, Gene Deletion, Microfilament Proteins, Models, Biological, Protein Binding, Protein Sorting Signals, Protein Structure, Tertiary, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Signal Transduction, Structure-Activity Relationship, Two-Hybrid System Techniques
Amino Acid Motifs, Amino Acid Sequence, Binding Sites, Carrier Proteins, Conserved Sequence, Cytoskeletal Proteins, Endocytosis, Fungal Proteins, Gene Deletion, Microfilament Proteins, Models, Biological, Protein Binding, Protein Sorting Signals, Protein Structure, Tertiary, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Signal Transduction, Structure-Activity Relationship, Two-Hybrid System Techniques
J. Cell Biol.
Date: Apr. 15, 2002
PubMed ID: 11940605
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