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Mutants in trs120 disrupt traffic from the early endosome to the late Golgi.

Cai H, Zhang Y, Pypaert M, Walker L, Ferro-Novick S - J. Cell Biol. (2005)

Bottom Line: Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II).Surprisingly, we report that mutations in trs120 do not block general secretion.Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06519, USA.

ABSTRACT
Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II). Both complexes share seven subunits, whereas three subunits (Trs130p, -120p, and -65p) are specific to TRAPPII. Previous studies have shown that mutations in the TRAPPII-specific gene trs130 block traffic through or from the Golgi. Surprisingly, we report that mutations in trs120 do not block general secretion. Instead, trs120 mutants accumulate aberrant membrane structures that resemble Berkeley bodies and disrupt the traffic of proteins that recycle through the early endosome. Mutants defective in recycling also display a defect in the localization of coat protein I (COPI) subunits, implying that Trs120p may participate in a COPI-dependent trafficking step on the early endosomal pathway. Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p. Our findings imply that Trs120p is required for vesicle traffic from the early endosome to the late Golgi.

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The trs120-2, -4, and -8 mutants display a defect in the localization of coatomer subunits. (A) Wild-type (wt) and mutant cells containing Sec21p-GFP, Sec7p-GFP, or Och1p-HA were grown to early log phase at 25°C and then shifted to 37°C for 15 min. For the analysis of Och1p-HA, cells were processed for immunofluorescence using a monoclonal anti-HA antibody. (B) Wild-type and mutant cells containing Ret2p-GFP were grown at 25°C and then shifted to 37°C for 15 min. (C) To immobilize TRAPP on beads, 50 mg of lysate prepared from a Bet3p PrA-tagged strain (lanes 2 and 3) was incubated with 30 μl of IgG–Sepharose beads for 2 h at 4°C. As a control, an untagged lysate (lane 1) was incubated with beads in the same way. The beads were washed three times with 1 ml of wash buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM DTT, 2.5 mM MgCl2, 1% Triton X-100, and 1× Protease inhibitor cocktail) and then incubated with 50 mg of wild type (lanes 1 and 2) or ret1-1 lysate (lane 3) for 3 h. After the incubation, the beads were washed four times with 1 ml of wash buffer, eluted with 0.2 M glycine, pH 2.8, and neutralized before loading onto an SDS–polyacrylamide gel. To detect Ret1p in lysates, equal amounts of wild-type (lane 4) and ret1-1 (lane 5) lysates were subjected to SDS gel electrophoresis and blotted with anti-coatomer antibody. (D) Lysates prepared from a Ret1p TAP-tagged (lane 2) or untagged (lane 1) strain were incubated with 30 μl of IgG–Sepharose beads for 2–3 h at 4°C. The beads were washed and treated as described in C. The lysate containing TAP-tagged Ret1p (lane 3) was subjected to SDS-PAGE and blotted with anti-Trs33p and anti-Sec13p antibodies.
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fig10: The trs120-2, -4, and -8 mutants display a defect in the localization of coatomer subunits. (A) Wild-type (wt) and mutant cells containing Sec21p-GFP, Sec7p-GFP, or Och1p-HA were grown to early log phase at 25°C and then shifted to 37°C for 15 min. For the analysis of Och1p-HA, cells were processed for immunofluorescence using a monoclonal anti-HA antibody. (B) Wild-type and mutant cells containing Ret2p-GFP were grown at 25°C and then shifted to 37°C for 15 min. (C) To immobilize TRAPP on beads, 50 mg of lysate prepared from a Bet3p PrA-tagged strain (lanes 2 and 3) was incubated with 30 μl of IgG–Sepharose beads for 2 h at 4°C. As a control, an untagged lysate (lane 1) was incubated with beads in the same way. The beads were washed three times with 1 ml of wash buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM DTT, 2.5 mM MgCl2, 1% Triton X-100, and 1× Protease inhibitor cocktail) and then incubated with 50 mg of wild type (lanes 1 and 2) or ret1-1 lysate (lane 3) for 3 h. After the incubation, the beads were washed four times with 1 ml of wash buffer, eluted with 0.2 M glycine, pH 2.8, and neutralized before loading onto an SDS–polyacrylamide gel. To detect Ret1p in lysates, equal amounts of wild-type (lane 4) and ret1-1 (lane 5) lysates were subjected to SDS gel electrophoresis and blotted with anti-coatomer antibody. (D) Lysates prepared from a Ret1p TAP-tagged (lane 2) or untagged (lane 1) strain were incubated with 30 μl of IgG–Sepharose beads for 2–3 h at 4°C. The beads were washed and treated as described in C. The lysate containing TAP-tagged Ret1p (lane 3) was subjected to SDS-PAGE and blotted with anti-Trs33p and anti-Sec13p antibodies.

Mentions: To monitor the localization of COPI, we used Sec21p-GFP, the γ subunit of COPI fused to GFP (Hosobuchi et al., 1992; Rossanese et al., 2001). In wild-type cells shifted to 37°C for 15 min, Sec21p-GFP localized to punctate structures in the cytoplasm (Fig. 10 A). Interestingly, three trs120 mutants (trs120-2, -4, and -8) displayed defects in the localization of Sec21p-GFP. Besides puncta, a uniform haze throughout the cytosol was also observed in these mutants (Fig. 10 A). The same result was obtained with permissively grown cells. In contrast, as shown in Fig. 10 A, trs130 mutants showed wild-type staining. The localization of a second coat subunit, Ret2p-GFP, was also defective in the same trs120 mutants (Fig. 10 B). The cytosolic haze observed with COPI subunits is not attributable to cleaved GFP, as none was detected by immunoblotting whole cell lysates with an anti-GFP antibody (unpublished data).


Mutants in trs120 disrupt traffic from the early endosome to the late Golgi.

Cai H, Zhang Y, Pypaert M, Walker L, Ferro-Novick S - J. Cell Biol. (2005)

The trs120-2, -4, and -8 mutants display a defect in the localization of coatomer subunits. (A) Wild-type (wt) and mutant cells containing Sec21p-GFP, Sec7p-GFP, or Och1p-HA were grown to early log phase at 25°C and then shifted to 37°C for 15 min. For the analysis of Och1p-HA, cells were processed for immunofluorescence using a monoclonal anti-HA antibody. (B) Wild-type and mutant cells containing Ret2p-GFP were grown at 25°C and then shifted to 37°C for 15 min. (C) To immobilize TRAPP on beads, 50 mg of lysate prepared from a Bet3p PrA-tagged strain (lanes 2 and 3) was incubated with 30 μl of IgG–Sepharose beads for 2 h at 4°C. As a control, an untagged lysate (lane 1) was incubated with beads in the same way. The beads were washed three times with 1 ml of wash buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM DTT, 2.5 mM MgCl2, 1% Triton X-100, and 1× Protease inhibitor cocktail) and then incubated with 50 mg of wild type (lanes 1 and 2) or ret1-1 lysate (lane 3) for 3 h. After the incubation, the beads were washed four times with 1 ml of wash buffer, eluted with 0.2 M glycine, pH 2.8, and neutralized before loading onto an SDS–polyacrylamide gel. To detect Ret1p in lysates, equal amounts of wild-type (lane 4) and ret1-1 (lane 5) lysates were subjected to SDS gel electrophoresis and blotted with anti-coatomer antibody. (D) Lysates prepared from a Ret1p TAP-tagged (lane 2) or untagged (lane 1) strain were incubated with 30 μl of IgG–Sepharose beads for 2–3 h at 4°C. The beads were washed and treated as described in C. The lysate containing TAP-tagged Ret1p (lane 3) was subjected to SDS-PAGE and blotted with anti-Trs33p and anti-Sec13p antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171297&req=5

fig10: The trs120-2, -4, and -8 mutants display a defect in the localization of coatomer subunits. (A) Wild-type (wt) and mutant cells containing Sec21p-GFP, Sec7p-GFP, or Och1p-HA were grown to early log phase at 25°C and then shifted to 37°C for 15 min. For the analysis of Och1p-HA, cells were processed for immunofluorescence using a monoclonal anti-HA antibody. (B) Wild-type and mutant cells containing Ret2p-GFP were grown at 25°C and then shifted to 37°C for 15 min. (C) To immobilize TRAPP on beads, 50 mg of lysate prepared from a Bet3p PrA-tagged strain (lanes 2 and 3) was incubated with 30 μl of IgG–Sepharose beads for 2 h at 4°C. As a control, an untagged lysate (lane 1) was incubated with beads in the same way. The beads were washed three times with 1 ml of wash buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM DTT, 2.5 mM MgCl2, 1% Triton X-100, and 1× Protease inhibitor cocktail) and then incubated with 50 mg of wild type (lanes 1 and 2) or ret1-1 lysate (lane 3) for 3 h. After the incubation, the beads were washed four times with 1 ml of wash buffer, eluted with 0.2 M glycine, pH 2.8, and neutralized before loading onto an SDS–polyacrylamide gel. To detect Ret1p in lysates, equal amounts of wild-type (lane 4) and ret1-1 (lane 5) lysates were subjected to SDS gel electrophoresis and blotted with anti-coatomer antibody. (D) Lysates prepared from a Ret1p TAP-tagged (lane 2) or untagged (lane 1) strain were incubated with 30 μl of IgG–Sepharose beads for 2–3 h at 4°C. The beads were washed and treated as described in C. The lysate containing TAP-tagged Ret1p (lane 3) was subjected to SDS-PAGE and blotted with anti-Trs33p and anti-Sec13p antibodies.
Mentions: To monitor the localization of COPI, we used Sec21p-GFP, the γ subunit of COPI fused to GFP (Hosobuchi et al., 1992; Rossanese et al., 2001). In wild-type cells shifted to 37°C for 15 min, Sec21p-GFP localized to punctate structures in the cytoplasm (Fig. 10 A). Interestingly, three trs120 mutants (trs120-2, -4, and -8) displayed defects in the localization of Sec21p-GFP. Besides puncta, a uniform haze throughout the cytosol was also observed in these mutants (Fig. 10 A). The same result was obtained with permissively grown cells. In contrast, as shown in Fig. 10 A, trs130 mutants showed wild-type staining. The localization of a second coat subunit, Ret2p-GFP, was also defective in the same trs120 mutants (Fig. 10 B). The cytosolic haze observed with COPI subunits is not attributable to cleaved GFP, as none was detected by immunoblotting whole cell lysates with an anti-GFP antibody (unpublished data).

Bottom Line: Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II).Surprisingly, we report that mutations in trs120 do not block general secretion.Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06519, USA.

ABSTRACT
Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II). Both complexes share seven subunits, whereas three subunits (Trs130p, -120p, and -65p) are specific to TRAPPII. Previous studies have shown that mutations in the TRAPPII-specific gene trs130 block traffic through or from the Golgi. Surprisingly, we report that mutations in trs120 do not block general secretion. Instead, trs120 mutants accumulate aberrant membrane structures that resemble Berkeley bodies and disrupt the traffic of proteins that recycle through the early endosome. Mutants defective in recycling also display a defect in the localization of coat protein I (COPI) subunits, implying that Trs120p may participate in a COPI-dependent trafficking step on the early endosomal pathway. Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p. Our findings imply that Trs120p is required for vesicle traffic from the early endosome to the late Golgi.

Show MeSH
Related in: MedlinePlus