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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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NPFSD interacts specifically with the SHD1 region of Sla1p. (A) Two-hybrid analysis of the NPFSD-interacting region of Sla1p. Fragments encoded by the original SLA1 clone isolated in the two-hybrid screen (471–1,185) and two deletion mutants are diagrammed. Numbers indicate the amino acid boundaries of Sla1p fused to the Gal4p activation domain. Each construct was tested for interaction with active (NPF) and inactive (NPA) versions of the Kex2p-derived signal by measuring β-galactosidase activity (units × 103) in cell extracts. (B) GST-fusion protein affinity chromatography of SHD1 from cell extracts. Triple repeats of active (NPF) and inactive (NPA) forms of the 11 aa Kex2p-derived signal joined to Ste2p residues 298–318 were fused to GST and expressed in E. coli. Fusion proteins bound to glutathione-Sepharose were incubated with extracts from E. coli cells expressing hexahistidine-tagged SHD1. Bound proteins were eluted with reduced glutathione, separated by SDS-PAGE, and visualized by staining with Coomassie blue or by immunoblotting with antibodies against hexahistidine epitope. Positions of molecular mass standards (in kD), GST fusions, SHD1, and an apparently dimeric form of SHD1 (*) are indicated. Input represents 0.5% of the extract used for the incubations with GST fusions. (C) SHD1 purified as described in Materials and methods was tested for interactions with GST-NPASD (NPA) or GST-NPFSD (NPF) as described in B. Eluted proteins were subjected to SDS-PAGE and detected by Coomassie blue staining. Input represents 30% of purified SHD1 incubated with GST fusions.
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fig5: NPFSD interacts specifically with the SHD1 region of Sla1p. (A) Two-hybrid analysis of the NPFSD-interacting region of Sla1p. Fragments encoded by the original SLA1 clone isolated in the two-hybrid screen (471–1,185) and two deletion mutants are diagrammed. Numbers indicate the amino acid boundaries of Sla1p fused to the Gal4p activation domain. Each construct was tested for interaction with active (NPF) and inactive (NPA) versions of the Kex2p-derived signal by measuring β-galactosidase activity (units × 103) in cell extracts. (B) GST-fusion protein affinity chromatography of SHD1 from cell extracts. Triple repeats of active (NPF) and inactive (NPA) forms of the 11 aa Kex2p-derived signal joined to Ste2p residues 298–318 were fused to GST and expressed in E. coli. Fusion proteins bound to glutathione-Sepharose were incubated with extracts from E. coli cells expressing hexahistidine-tagged SHD1. Bound proteins were eluted with reduced glutathione, separated by SDS-PAGE, and visualized by staining with Coomassie blue or by immunoblotting with antibodies against hexahistidine epitope. Positions of molecular mass standards (in kD), GST fusions, SHD1, and an apparently dimeric form of SHD1 (*) are indicated. Input represents 0.5% of the extract used for the incubations with GST fusions. (C) SHD1 purified as described in Materials and methods was tested for interactions with GST-NPASD (NPA) or GST-NPFSD (NPF) as described in B. Eluted proteins were subjected to SDS-PAGE and detected by Coomassie blue staining. Input represents 30% of purified SHD1 incubated with GST fusions.

Mentions: To define the region of Sla1p that interacts with NPFSD, deletion mutants of the SLA1 fragment were tested for NPFSD binding by two-hybrid analysis. The fragment isolated in the original two-hybrid screen extended from K471 to T1185, encompassing the two SHD domains, the proline-rich sequence, and a majority of the COOH-terminal TGGXXXPQ repeats (Fig. 5 A). Using β-galactosidase activity as a measure of interaction, COOH-terminal excision of most of the fragment, leaving only K471-E555, produced little effect on NPFSD interaction (Fig. 5 A). In contrast, an NH2-terminal truncation of 39 aa reduced β-galactosidase activity to ∼10% of the original fragment (Fig. 5 A). These results suggest that the 84 aa between K471 and E555 contain an NPFX(1,2)D interaction site. This fragment includes SHD1 (P492-G551), first recognized as a domain of unknown function that is conserved in an orthologue of SLA1 from Schizosaccharomyces pombe (Ayscough et al., 1999). (It should be noted that SHD1 is distinct from a region in Pan1p containing TGGXXXPQ repeats that has been referred to as a Sla1p homology domain [Tang and Cai, 1996].)


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 interacts specifically with the SHD1 region of Sla1p. (A) Two-hybrid analysis of the NPFSD-interacting region of Sla1p. Fragments encoded by the original SLA1 clone isolated in the two-hybrid screen (471–1,185) and two deletion mutants are diagrammed. Numbers indicate the amino acid boundaries of Sla1p fused to the Gal4p activation domain. Each construct was tested for interaction with active (NPF) and inactive (NPA) versions of the Kex2p-derived signal by measuring β-galactosidase activity (units × 103) in cell extracts. (B) GST-fusion protein affinity chromatography of SHD1 from cell extracts. Triple repeats of active (NPF) and inactive (NPA) forms of the 11 aa Kex2p-derived signal joined to Ste2p residues 298–318 were fused to GST and expressed in E. coli. Fusion proteins bound to glutathione-Sepharose were incubated with extracts from E. coli cells expressing hexahistidine-tagged SHD1. Bound proteins were eluted with reduced glutathione, separated by SDS-PAGE, and visualized by staining with Coomassie blue or by immunoblotting with antibodies against hexahistidine epitope. Positions of molecular mass standards (in kD), GST fusions, SHD1, and an apparently dimeric form of SHD1 (*) are indicated. Input represents 0.5% of the extract used for the incubations with GST fusions. (C) SHD1 purified as described in Materials and methods was tested for interactions with GST-NPASD (NPA) or GST-NPFSD (NPF) as described in B. Eluted proteins were subjected to SDS-PAGE and detected by Coomassie blue staining. Input represents 30% of purified SHD1 incubated with GST fusions.
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fig5: NPFSD interacts specifically with the SHD1 region of Sla1p. (A) Two-hybrid analysis of the NPFSD-interacting region of Sla1p. Fragments encoded by the original SLA1 clone isolated in the two-hybrid screen (471–1,185) and two deletion mutants are diagrammed. Numbers indicate the amino acid boundaries of Sla1p fused to the Gal4p activation domain. Each construct was tested for interaction with active (NPF) and inactive (NPA) versions of the Kex2p-derived signal by measuring β-galactosidase activity (units × 103) in cell extracts. (B) GST-fusion protein affinity chromatography of SHD1 from cell extracts. Triple repeats of active (NPF) and inactive (NPA) forms of the 11 aa Kex2p-derived signal joined to Ste2p residues 298–318 were fused to GST and expressed in E. coli. Fusion proteins bound to glutathione-Sepharose were incubated with extracts from E. coli cells expressing hexahistidine-tagged SHD1. Bound proteins were eluted with reduced glutathione, separated by SDS-PAGE, and visualized by staining with Coomassie blue or by immunoblotting with antibodies against hexahistidine epitope. Positions of molecular mass standards (in kD), GST fusions, SHD1, and an apparently dimeric form of SHD1 (*) are indicated. Input represents 0.5% of the extract used for the incubations with GST fusions. (C) SHD1 purified as described in Materials and methods was tested for interactions with GST-NPASD (NPA) or GST-NPFSD (NPF) as described in B. Eluted proteins were subjected to SDS-PAGE and detected by Coomassie blue staining. Input represents 30% of purified SHD1 incubated with GST fusions.
Mentions: To define the region of Sla1p that interacts with NPFSD, deletion mutants of the SLA1 fragment were tested for NPFSD binding by two-hybrid analysis. The fragment isolated in the original two-hybrid screen extended from K471 to T1185, encompassing the two SHD domains, the proline-rich sequence, and a majority of the COOH-terminal TGGXXXPQ repeats (Fig. 5 A). Using β-galactosidase activity as a measure of interaction, COOH-terminal excision of most of the fragment, leaving only K471-E555, produced little effect on NPFSD interaction (Fig. 5 A). In contrast, an NH2-terminal truncation of 39 aa reduced β-galactosidase activity to ∼10% of the original fragment (Fig. 5 A). These results suggest that the 84 aa between K471 and E555 contain an NPFX(1,2)D interaction site. This fragment includes SHD1 (P492-G551), first recognized as a domain of unknown function that is conserved in an orthologue of SLA1 from Schizosaccharomyces pombe (Ayscough et al., 1999). (It should be noted that SHD1 is distinct from a region in Pan1p containing TGGXXXPQ repeats that has been referred to as a Sla1p homology domain [Tang and Cai, 1996].)

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