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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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NPFX(1,2)D-mediated endocytosis requires SLA1. (A and B) Centromere plasmids encoding Ste2p-G392N, Ste2p-NPFXD (Ste2p11KR-G392N), and Ste2p-GPFXD (Ste2p11KR) were introduced into SLA1 cells (GPY2490, GPY2454, and GPY2665), and the same plasmids plus Ste2p-Ub (Ste2p-G392A) were introduced into sla1Δ cells (GPY2487, GPY 2488, GPY2489, GPY2451, and GPY2666). Endocytosis was monitored by α-factor uptake as described in the legend to Fig. 1.
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fig4: NPFX(1,2)D-mediated endocytosis requires SLA1. (A and B) Centromere plasmids encoding Ste2p-G392N, Ste2p-NPFXD (Ste2p11KR-G392N), and Ste2p-GPFXD (Ste2p11KR) were introduced into SLA1 cells (GPY2490, GPY2454, and GPY2665), and the same plasmids plus Ste2p-Ub (Ste2p-G392A) were introduced into sla1Δ cells (GPY2487, GPY 2488, GPY2489, GPY2451, and GPY2666). Endocytosis was monitored by α-factor uptake as described in the legend to Fig. 1.

Mentions: The role of Sla1p in NPFX(1,2)D-mediated endocytosis was investigated by evaluating the effects of disrupting SLA1 (sla1Δ) on internalization of Ste2p-G392N, Ste2p-NPFXD (Ste2p11KR-G392N), and Ste2p-Ub (Ste2p-G392A). Uptake of α-factor mediated by Ste2p-NPFXD was abolished in sla1Δ cells (Fig. 4 A). The effects of sla1Δ on Ste2p-G392N and Ste2p-Ub were far more subtle; after a 5-min lag, internalization of both receptors was essentially normal. Sla1p was also required for uptake directed by the native GPFAD sequence in Ste2p (Fig. 4 B). The selectivity of sla1Δ effects on Ste2p-NPFXD and Ste2p-GPFXD supports the hypothesis that Sla1p acts as a specific endocytic adaptor for the NPFX(1,2)D signal. However, the lag in ubiquitin-mediated endocytosis suggests Sla1p also participates more generally in the initiation of cargo internalization.


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)

NPFX(1,2)D-mediated endocytosis requires SLA1. (A and B) Centromere plasmids encoding Ste2p-G392N, Ste2p-NPFXD (Ste2p11KR-G392N), and Ste2p-GPFXD (Ste2p11KR) were introduced into SLA1 cells (GPY2490, GPY2454, and GPY2665), and the same plasmids plus Ste2p-Ub (Ste2p-G392A) were introduced into sla1Δ cells (GPY2487, GPY 2488, GPY2489, GPY2451, and GPY2666). Endocytosis was monitored by α-factor uptake as described in the legend to Fig. 1.
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Related In: Results  -  Collection

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fig4: NPFX(1,2)D-mediated endocytosis requires SLA1. (A and B) Centromere plasmids encoding Ste2p-G392N, Ste2p-NPFXD (Ste2p11KR-G392N), and Ste2p-GPFXD (Ste2p11KR) were introduced into SLA1 cells (GPY2490, GPY2454, and GPY2665), and the same plasmids plus Ste2p-Ub (Ste2p-G392A) were introduced into sla1Δ cells (GPY2487, GPY 2488, GPY2489, GPY2451, and GPY2666). Endocytosis was monitored by α-factor uptake as described in the legend to Fig. 1.
Mentions: The role of Sla1p in NPFX(1,2)D-mediated endocytosis was investigated by evaluating the effects of disrupting SLA1 (sla1Δ) on internalization of Ste2p-G392N, Ste2p-NPFXD (Ste2p11KR-G392N), and Ste2p-Ub (Ste2p-G392A). Uptake of α-factor mediated by Ste2p-NPFXD was abolished in sla1Δ cells (Fig. 4 A). The effects of sla1Δ on Ste2p-G392N and Ste2p-Ub were far more subtle; after a 5-min lag, internalization of both receptors was essentially normal. Sla1p was also required for uptake directed by the native GPFAD sequence in Ste2p (Fig. 4 B). The selectivity of sla1Δ effects on Ste2p-NPFXD and Ste2p-GPFXD supports the hypothesis that Sla1p acts as a specific endocytic adaptor for the NPFX(1,2)D signal. However, the lag in ubiquitin-mediated endocytosis suggests Sla1p also participates more generally in the initiation of cargo internalization.

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