Limits...
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.

Show MeSH

Related in: MedlinePlus

NPFSD specifically interacts with the COOH-terminal region of Sla1p. (A) Bait constructs used in the two-hybrid analysis. Three repeats of the NPFSD signal linked by Ste2p aa298–318 to the Gal4p DNA binding domain (GBD), and mutant versions shown below, were tested for interaction with the SLA1 fragment isolated in the two-hybrid screen. Interaction was assessed in PJ69–4A by growth of serial diluted cells at 30°C on synthetic media containing (+ADE) or lacking (−ADE) adenine. (B) Domain architecture of Sla1p. Indicated are SH3 domains, SLA1 homology domain 1 and 2 (SHD1 and SHD2), a predicted proline-rich SH3 binding site (P), and multiple repeats of TGGXXXPQ (vertical lines). The Sla1p fragment isolated from the two-hybrid screen (K471-T1185) is underlined. (C) The Sla1p fragment and indicated EH-domain proteins were tested for interaction with NPFSD as in A. (D) Yeast EH-domain encoding genes were tested for interaction with YAP180s as above.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199253&req=5

fig3: NPFSD specifically interacts with the COOH-terminal region of Sla1p. (A) Bait constructs used in the two-hybrid analysis. Three repeats of the NPFSD signal linked by Ste2p aa298–318 to the Gal4p DNA binding domain (GBD), and mutant versions shown below, were tested for interaction with the SLA1 fragment isolated in the two-hybrid screen. Interaction was assessed in PJ69–4A by growth of serial diluted cells at 30°C on synthetic media containing (+ADE) or lacking (−ADE) adenine. (B) Domain architecture of Sla1p. Indicated are SH3 domains, SLA1 homology domain 1 and 2 (SHD1 and SHD2), a predicted proline-rich SH3 binding site (P), and multiple repeats of TGGXXXPQ (vertical lines). The Sla1p fragment isolated from the two-hybrid screen (K471-T1185) is underlined. (C) The Sla1p fragment and indicated EH-domain proteins were tested for interaction with NPFSD as in A. (D) Yeast EH-domain encoding genes were tested for interaction with YAP180s as above.

Mentions: To isolate an endocytic adaptor for the NPFX(1,2)D signal, we carried out a yeast two-hybrid screen. We generated a bait that carried the cytoplasmic domain sequences from Ste2pΔ318 fused to three tandem copies of the 11-residue Kex2p-derived NPFSD sequence (Fig. 3 A). To eliminate activation of reporter genes by the NPFSD bait alone, glutamates in the Kex2p derived sequences were altered to alanine, a mutation that does not affect NPFX(1,2)D-mediated endocytosis (Tan et al., 1996). We reasoned that the triple repeat might improve chances of detecting interactions, as the initial internalization rate of Ste2pΔ318-NPFSD was enhanced ∼2.5-fold by adding two additional copies of the Kex2p NPFSD-containing sequence (unpublished data). Using the triple-repeat bait we identified a plasmid carrying a fragment of the SLA1 gene, but no plasmids expressing EH domain proteins. SLA1 encodes a 1,244-aa protein implicated in actin dynamics (Holtzman et al., 1993; Ayscough et al., 1997, 1999). Furthermore, Sla1p interacts with Pan1p and End3p, two proteins that function in actin organization and endocytosis, suggesting a role for Sla1p in the endocytic process (Tang et al., 2000). Sla1p contains three NH2-terminal SH3 domains, a central region with a proline rich sequence flanked by two domains unrelated to each other but conserved in other fungal and plant species (SLA1 homology domains [SHDs]), and multiple repeats of the sequence TGGXXXPQ in the COOH-terminal domain that potentially serve as phosphorylation sites (Fig. 3 B) (Ayscough et al., 1999; Zeng and Cai, 1999). The fragment isolated in the two-hybrid screen spans aa 471–1185, lacking the SH3 domains.


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)

NPFSD specifically interacts with the COOH-terminal region of Sla1p. (A) Bait constructs used in the two-hybrid analysis. Three repeats of the NPFSD signal linked by Ste2p aa298–318 to the Gal4p DNA binding domain (GBD), and mutant versions shown below, were tested for interaction with the SLA1 fragment isolated in the two-hybrid screen. Interaction was assessed in PJ69–4A by growth of serial diluted cells at 30°C on synthetic media containing (+ADE) or lacking (−ADE) adenine. (B) Domain architecture of Sla1p. Indicated are SH3 domains, SLA1 homology domain 1 and 2 (SHD1 and SHD2), a predicted proline-rich SH3 binding site (P), and multiple repeats of TGGXXXPQ (vertical lines). The Sla1p fragment isolated from the two-hybrid screen (K471-T1185) is underlined. (C) The Sla1p fragment and indicated EH-domain proteins were tested for interaction with NPFSD as in A. (D) Yeast EH-domain encoding genes were tested for interaction with YAP180s as above.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: NPFSD specifically interacts with the COOH-terminal region of Sla1p. (A) Bait constructs used in the two-hybrid analysis. Three repeats of the NPFSD signal linked by Ste2p aa298–318 to the Gal4p DNA binding domain (GBD), and mutant versions shown below, were tested for interaction with the SLA1 fragment isolated in the two-hybrid screen. Interaction was assessed in PJ69–4A by growth of serial diluted cells at 30°C on synthetic media containing (+ADE) or lacking (−ADE) adenine. (B) Domain architecture of Sla1p. Indicated are SH3 domains, SLA1 homology domain 1 and 2 (SHD1 and SHD2), a predicted proline-rich SH3 binding site (P), and multiple repeats of TGGXXXPQ (vertical lines). The Sla1p fragment isolated from the two-hybrid screen (K471-T1185) is underlined. (C) The Sla1p fragment and indicated EH-domain proteins were tested for interaction with NPFSD as in A. (D) Yeast EH-domain encoding genes were tested for interaction with YAP180s as above.
Mentions: To isolate an endocytic adaptor for the NPFX(1,2)D signal, we carried out a yeast two-hybrid screen. We generated a bait that carried the cytoplasmic domain sequences from Ste2pΔ318 fused to three tandem copies of the 11-residue Kex2p-derived NPFSD sequence (Fig. 3 A). To eliminate activation of reporter genes by the NPFSD bait alone, glutamates in the Kex2p derived sequences were altered to alanine, a mutation that does not affect NPFX(1,2)D-mediated endocytosis (Tan et al., 1996). We reasoned that the triple repeat might improve chances of detecting interactions, as the initial internalization rate of Ste2pΔ318-NPFSD was enhanced ∼2.5-fold by adding two additional copies of the Kex2p NPFSD-containing sequence (unpublished data). Using the triple-repeat bait we identified a plasmid carrying a fragment of the SLA1 gene, but no plasmids expressing EH domain proteins. SLA1 encodes a 1,244-aa protein implicated in actin dynamics (Holtzman et al., 1993; Ayscough et al., 1997, 1999). Furthermore, Sla1p interacts with Pan1p and End3p, two proteins that function in actin organization and endocytosis, suggesting a role for Sla1p in the endocytic process (Tang et al., 2000). Sla1p contains three NH2-terminal SH3 domains, a central region with a proline rich sequence flanked by two domains unrelated to each other but conserved in other fungal and plant species (SLA1 homology domains [SHDs]), and multiple repeats of the sequence TGGXXXPQ in the COOH-terminal domain that potentially serve as phosphorylation sites (Fig. 3 B) (Ayscough et al., 1999; Zeng and Cai, 1999). The fragment isolated in the two-hybrid screen spans aa 471–1185, lacking the SH3 domains.

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