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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 SHD1. (A) Effects of deleting Sla1p SHD1 on receptor mediated endocytosis. Uptake of Ste2p-G392N, Ste2p-Ub (Ste2p-G392A), or Ste2p-NPFXD (Ste2p11KR-G392N) was measured in sla1-Δshd1 cells (GPY 2493, GPY2494, and GPY 2495) and Ste2p-G392N in SLA1 cells (GPY2490) as in Fig. 1. (B) Effects of deleting Sla1p SHD1 on growth. GPY2448 (sla1Δ) was transformed with centromeric plasmids containing no insert (vector), wild-type SLA1, or sla1-Δshd1 and the resulting strains (GPY2487, GPY 2490, and GPY 2493, respectively) plus wild-type strain GPY1805 were serially diluted onto synthetic media and examined for growth after 3 d at the indicated temperatures. (C) Effects of deleting Sla1p SHD1 on actin organization. Gallery of cells from strains GPY2487 (sla1Δ, vector), GPY2490 (sla1Δ, SLA1), and GPY2493 (sla1Δ, sla1-Δshd1) grown at 30°C and then fixed and stained with rhodamine phalloidin. Bar, 5 microns.
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fig6: NPFX(1,2)D mediated endocytosis requires SHD1. (A) Effects of deleting Sla1p SHD1 on receptor mediated endocytosis. Uptake of Ste2p-G392N, Ste2p-Ub (Ste2p-G392A), or Ste2p-NPFXD (Ste2p11KR-G392N) was measured in sla1-Δshd1 cells (GPY 2493, GPY2494, and GPY 2495) and Ste2p-G392N in SLA1 cells (GPY2490) as in Fig. 1. (B) Effects of deleting Sla1p SHD1 on growth. GPY2448 (sla1Δ) was transformed with centromeric plasmids containing no insert (vector), wild-type SLA1, or sla1-Δshd1 and the resulting strains (GPY2487, GPY 2490, and GPY 2493, respectively) plus wild-type strain GPY1805 were serially diluted onto synthetic media and examined for growth after 3 d at the indicated temperatures. (C) Effects of deleting Sla1p SHD1 on actin organization. Gallery of cells from strains GPY2487 (sla1Δ, vector), GPY2490 (sla1Δ, SLA1), and GPY2493 (sla1Δ, sla1-Δshd1) grown at 30°C and then fixed and stained with rhodamine phalloidin. Bar, 5 microns.

Mentions: We designed a mutant of SLA1 lacking sequences encoding SHD1 (P492-G551, sla1-Δshd1) to examine the role of this domain in endocytosis. Centromere-based plasmids expressing either SLA1 or sla1-Δshd1 were introduced into sla1Δ cells and Sla1p expression was monitored by immunoblotting. The results indicated that deletion of SHD1 does not destabilize Sla1p (unpublished data). We then compared endocytosis of Ste2p-G392N, Ste2p-NPFXD, and Ste2p-Ub in sla1-Δshd1 cells to Ste2p-G392N endocytosis in wild-type cells. As shown in Fig. 6 A, internalization of Ste2p-G392N and Ste2p-Ub in sla1-Δshd1 cells lagged only at the 5-min time point compared to the wild-type control. The delay was less pronounced than that observed for the same receptors in sla1Δ cells (compare Figs. 4 and 6 A). However, uptake of Ste2p-NPFXD was essentially eliminated (Fig. 6 A), as was uptake of Ste2p-GPFXD (unpublished data), equivalent to the effects of deleting the entire SLA1 gene (Fig. 4).


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 SHD1. (A) Effects of deleting Sla1p SHD1 on receptor mediated endocytosis. Uptake of Ste2p-G392N, Ste2p-Ub (Ste2p-G392A), or Ste2p-NPFXD (Ste2p11KR-G392N) was measured in sla1-Δshd1 cells (GPY 2493, GPY2494, and GPY 2495) and Ste2p-G392N in SLA1 cells (GPY2490) as in Fig. 1. (B) Effects of deleting Sla1p SHD1 on growth. GPY2448 (sla1Δ) was transformed with centromeric plasmids containing no insert (vector), wild-type SLA1, or sla1-Δshd1 and the resulting strains (GPY2487, GPY 2490, and GPY 2493, respectively) plus wild-type strain GPY1805 were serially diluted onto synthetic media and examined for growth after 3 d at the indicated temperatures. (C) Effects of deleting Sla1p SHD1 on actin organization. Gallery of cells from strains GPY2487 (sla1Δ, vector), GPY2490 (sla1Δ, SLA1), and GPY2493 (sla1Δ, sla1-Δshd1) grown at 30°C and then fixed and stained with rhodamine phalloidin. Bar, 5 microns.
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fig6: NPFX(1,2)D mediated endocytosis requires SHD1. (A) Effects of deleting Sla1p SHD1 on receptor mediated endocytosis. Uptake of Ste2p-G392N, Ste2p-Ub (Ste2p-G392A), or Ste2p-NPFXD (Ste2p11KR-G392N) was measured in sla1-Δshd1 cells (GPY 2493, GPY2494, and GPY 2495) and Ste2p-G392N in SLA1 cells (GPY2490) as in Fig. 1. (B) Effects of deleting Sla1p SHD1 on growth. GPY2448 (sla1Δ) was transformed with centromeric plasmids containing no insert (vector), wild-type SLA1, or sla1-Δshd1 and the resulting strains (GPY2487, GPY 2490, and GPY 2493, respectively) plus wild-type strain GPY1805 were serially diluted onto synthetic media and examined for growth after 3 d at the indicated temperatures. (C) Effects of deleting Sla1p SHD1 on actin organization. Gallery of cells from strains GPY2487 (sla1Δ, vector), GPY2490 (sla1Δ, SLA1), and GPY2493 (sla1Δ, sla1-Δshd1) grown at 30°C and then fixed and stained with rhodamine phalloidin. Bar, 5 microns.
Mentions: We designed a mutant of SLA1 lacking sequences encoding SHD1 (P492-G551, sla1-Δshd1) to examine the role of this domain in endocytosis. Centromere-based plasmids expressing either SLA1 or sla1-Δshd1 were introduced into sla1Δ cells and Sla1p expression was monitored by immunoblotting. The results indicated that deletion of SHD1 does not destabilize Sla1p (unpublished data). We then compared endocytosis of Ste2p-G392N, Ste2p-NPFXD, and Ste2p-Ub in sla1-Δshd1 cells to Ste2p-G392N endocytosis in wild-type cells. As shown in Fig. 6 A, internalization of Ste2p-G392N and Ste2p-Ub in sla1-Δshd1 cells lagged only at the 5-min time point compared to the wild-type control. The delay was less pronounced than that observed for the same receptors in sla1Δ cells (compare Figs. 4 and 6 A). However, uptake of Ste2p-NPFXD was essentially eliminated (Fig. 6 A), as was uptake of Ste2p-GPFXD (unpublished data), equivalent to the effects of deleting the entire SLA1 gene (Fig. 4).

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