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

Howard JP, Hutton JL, Olson JM, Payne GS - J. Cell Biol. (2002)

Bottom Line: 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.Furthermore, EH domains did not recognize an NPFX(1,2)D signal when directly tested by two-hybrid analysis.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.

ABSTRACT
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.

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Model for Sla1p/Pan1p/End3p endocytic adaptor complex. Solid arrows indicate established physical interactions, dotted arrows indicate possible interactions. See text for details.
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fig7: Model for Sla1p/Pan1p/End3p endocytic adaptor complex. Solid arrows indicate established physical interactions, dotted arrows indicate possible interactions. See text for details.

Mentions: SLA1 was originally uncovered in a screen for mutations that result in lethality when combined with a deletion of the gene encoding the actin binding protein Abp1p (Holtzman et al., 1993). Characterization of sla1 mutants and Sla1p interaction partners indicate a role for Sla1p in actin cytoskeleton dynamics (Ayscough et al., 1997, 1999; Li, 1997; Madania et al., 1999; Drees et al., 2001). Importantly, Tang et al. (2000) have provided evidence that Sla1p forms a complex with Pan1p and End3p, two proteins essential for endocytosis. Our results extend the connection of Sla1p to endocytosis by demonstrating the role of the SHD1 domain in NPFX(1,2)D-mediated internalization. Considering these findings, we propose that the Sla1p/Pan1p/End3p complex serves as an endocytic adaptor complex linking cargo to the actin and clathrin-based endocytic machinery in yeast (Fig. 7).


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

Howard JP, Hutton JL, Olson JM, Payne GS - J. Cell Biol. (2002)

Model for Sla1p/Pan1p/End3p endocytic adaptor complex. Solid arrows indicate established physical interactions, dotted arrows indicate possible interactions. See text for details.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199253&req=5

fig7: Model for Sla1p/Pan1p/End3p endocytic adaptor complex. Solid arrows indicate established physical interactions, dotted arrows indicate possible interactions. See text for details.
Mentions: SLA1 was originally uncovered in a screen for mutations that result in lethality when combined with a deletion of the gene encoding the actin binding protein Abp1p (Holtzman et al., 1993). Characterization of sla1 mutants and Sla1p interaction partners indicate a role for Sla1p in actin cytoskeleton dynamics (Ayscough et al., 1997, 1999; Li, 1997; Madania et al., 1999; Drees et al., 2001). Importantly, Tang et al. (2000) have provided evidence that Sla1p forms a complex with Pan1p and End3p, two proteins essential for endocytosis. Our results extend the connection of Sla1p to endocytosis by demonstrating the role of the SHD1 domain in NPFX(1,2)D-mediated internalization. Considering these findings, we propose that the Sla1p/Pan1p/End3p complex serves as an endocytic adaptor complex linking cargo to the actin and clathrin-based endocytic machinery in yeast (Fig. 7).

Bottom Line: 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.Furthermore, EH domains did not recognize an NPFX(1,2)D signal when directly tested by two-hybrid analysis.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus