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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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Sequence requirements for NPFX(1,2)D-mediated internalization. (A) Diagrams of Ste2pΔ318-NPFSD and Ste2pΔ318*. The indicated sequences from Kex2p were fused to Ste2p at aa 318. Shaded rectangles represent the COOH-terminal region of Ste2p to aa 318 and the vertical black line represents the plasma membrane. The cytosolic tail domain of Ste2p is predicted to begin at aa 298. Residues previously defined as important for endocytic targeting by this signal are indicated by larger font. (B and C) Uptake of prebound, radiolabeled α-factor was measured in GPY779 expressing wild-type and indicated mutant forms of Ste2pΔ318-NPFSD at 30°C. Mutant residues are indicated in lowercase font. Error bars represent the standard deviation in three separate experiments.
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fig1: Sequence requirements for NPFX(1,2)D-mediated internalization. (A) Diagrams of Ste2pΔ318-NPFSD and Ste2pΔ318*. The indicated sequences from Kex2p were fused to Ste2p at aa 318. Shaded rectangles represent the COOH-terminal region of Ste2p to aa 318 and the vertical black line represents the plasma membrane. The cytosolic tail domain of Ste2p is predicted to begin at aa 298. Residues previously defined as important for endocytic targeting by this signal are indicated by larger font. (B and C) Uptake of prebound, radiolabeled α-factor was measured in GPY779 expressing wild-type and indicated mutant forms of Ste2pΔ318-NPFSD at 30°C. Mutant residues are indicated in lowercase font. Error bars represent the standard deviation in three separate experiments.

Mentions: NPFX(1,2)D was originally defined as an endocytic targeting signal by introducing alanine substitution mutations into a Ste2p/Kex2p chimera and a truncated form of Ste3p (Tan et al., 1996). However, in neither case was the targeting signal demonstrated to function completely independently of other Kex2p or Ste3p sequences. Consequently, an 11-aa sequence containing NPFSD from Kex2p was appended to residue 318 of Ste2p, replacing most of the Ste2p COOH-terminal cytoplasmic domain (Fig. 1 A, Ste2pΔ318-NPFSD). Truncation of Ste2p at position 318 leaves pheromone binding intact but eliminates the native endocytic targeting determinants (Konopka et al., 1988; Reneke et al., 1988). For comparison, a second construct was generated that carries the first five aa of the Kex2p sequence, but lacks the NPFSD signal (Fig. 1 A, Ste2pΔ318*). Ste2pΔ318-NPFSD and Ste2pΔ318* were introduced into cells lacking endogenous STE2, and endocytosis was assessed by measuring uptake of radiolabelled α-factor (Fig. 1, B and C). Ste2pΔ318-NPFSD was rapidly internalized (Fig. 1 C), albeit not as efficiently as wild-type Ste2p (compare Figs. 1 B and 2 A). In contrast, Ste2pΔ318* was not internalized (Fig. 1 C). These results indicate that the 11-aa peptide encompassing NPFSD functions autonomously to direct endocytosis in the absence of other Kex2p sequences.


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)

Sequence requirements for NPFX(1,2)D-mediated internalization. (A) Diagrams of Ste2pΔ318-NPFSD and Ste2pΔ318*. The indicated sequences from Kex2p were fused to Ste2p at aa 318. Shaded rectangles represent the COOH-terminal region of Ste2p to aa 318 and the vertical black line represents the plasma membrane. The cytosolic tail domain of Ste2p is predicted to begin at aa 298. Residues previously defined as important for endocytic targeting by this signal are indicated by larger font. (B and C) Uptake of prebound, radiolabeled α-factor was measured in GPY779 expressing wild-type and indicated mutant forms of Ste2pΔ318-NPFSD at 30°C. Mutant residues are indicated in lowercase font. Error bars represent the standard deviation in three separate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199253&req=5

fig1: Sequence requirements for NPFX(1,2)D-mediated internalization. (A) Diagrams of Ste2pΔ318-NPFSD and Ste2pΔ318*. The indicated sequences from Kex2p were fused to Ste2p at aa 318. Shaded rectangles represent the COOH-terminal region of Ste2p to aa 318 and the vertical black line represents the plasma membrane. The cytosolic tail domain of Ste2p is predicted to begin at aa 298. Residues previously defined as important for endocytic targeting by this signal are indicated by larger font. (B and C) Uptake of prebound, radiolabeled α-factor was measured in GPY779 expressing wild-type and indicated mutant forms of Ste2pΔ318-NPFSD at 30°C. Mutant residues are indicated in lowercase font. Error bars represent the standard deviation in three separate experiments.
Mentions: NPFX(1,2)D was originally defined as an endocytic targeting signal by introducing alanine substitution mutations into a Ste2p/Kex2p chimera and a truncated form of Ste3p (Tan et al., 1996). However, in neither case was the targeting signal demonstrated to function completely independently of other Kex2p or Ste3p sequences. Consequently, an 11-aa sequence containing NPFSD from Kex2p was appended to residue 318 of Ste2p, replacing most of the Ste2p COOH-terminal cytoplasmic domain (Fig. 1 A, Ste2pΔ318-NPFSD). Truncation of Ste2p at position 318 leaves pheromone binding intact but eliminates the native endocytic targeting determinants (Konopka et al., 1988; Reneke et al., 1988). For comparison, a second construct was generated that carries the first five aa of the Kex2p sequence, but lacks the NPFSD signal (Fig. 1 A, Ste2pΔ318*). Ste2pΔ318-NPFSD and Ste2pΔ318* were introduced into cells lacking endogenous STE2, and endocytosis was assessed by measuring uptake of radiolabelled α-factor (Fig. 1, B and C). Ste2pΔ318-NPFSD was rapidly internalized (Fig. 1 C), albeit not as efficiently as wild-type Ste2p (compare Figs. 1 B and 2 A). In contrast, Ste2pΔ318* was not internalized (Fig. 1 C). These results indicate that the 11-aa peptide encompassing NPFSD functions autonomously to direct endocytosis in the absence of other Kex2p sequences.

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