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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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TRAPPII largely colocalizes with Sec7p-GFP but not with PI(3)P-containing endosomal membranes. (A, top) Cells containing Sec7p-GFP and Trs120p-13myc were fixed and processed for immunofluorescence. (bottom) Cells containing Trs130p-GFP and Sec7p-DsRed were viewed by direct fluorescence microscopy. (B) Chs3p-GFP–containing puncta also largely colocalized with Sec7p-DsRed. Cells containing Sec7p-DsRed and Chs3p-GFP were viewed by direct fluorescence microscopy. (C) Cells containing Trs130p-GFP and DsRed-FYVE were viewed by direct fluorescence microscopy. Trs130p-GFP did not colocalize with DsRed-FYVE, a marker for PI(3)P-containing endosomal membranes.
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fig8: TRAPPII largely colocalizes with Sec7p-GFP but not with PI(3)P-containing endosomal membranes. (A, top) Cells containing Sec7p-GFP and Trs120p-13myc were fixed and processed for immunofluorescence. (bottom) Cells containing Trs130p-GFP and Sec7p-DsRed were viewed by direct fluorescence microscopy. (B) Chs3p-GFP–containing puncta also largely colocalized with Sec7p-DsRed. Cells containing Sec7p-DsRed and Chs3p-GFP were viewed by direct fluorescence microscopy. (C) Cells containing Trs130p-GFP and DsRed-FYVE were viewed by direct fluorescence microscopy. Trs130p-GFP did not colocalize with DsRed-FYVE, a marker for PI(3)P-containing endosomal membranes.

Mentions: Using a monoclonal anti-myc antibody, we performed indirect immunofluorescence to localize Trs120p-myc in cells containing the late Golgi marker Sec7p-GFP. Quantitation of 86 cells revealed that 314 of the 331 Trs120p-myc–containing puncta colocalized with Sec7p-GFP (Fig. 8 A, top). A total of 448 Sec7p-GFP–containing puncta were identified in the 86 cells that were quantitated, and 314 of these puncta colocalized with Trs120p-myc. Similar results were obtained when we examined the localization of Trs130p fused to 3-x-GFP with Sec7p-DsRed (Fig. 8 A, bottom). Furthermore, quantitation of >280 puncta revealed that ∼50% of the Sec7p-DsRed puncta colocalized with Chs3p-GFP and ∼60% of the Chs3p-GFP puncta colocalized with Sec7p-DsRed (Fig. 8 B). Thus, Chs3p and Sec7p largely colocalize with each by direct fluorescence. The localization of TRAPPII was also monitored with respect to the FYVE domain of early endosomal antigen 1, which marks phosphatidylinositol-3-phosphate (PI[3]P)–containing endosomal membranes (Burd and Emr, 1998; Gaullier et al., 1998; Katzmann et al., 2003). Trs130p–3-x-GFP did not colocalize with DsRed-FYVE, which was found on punctate structures adjacent to the vacuole (Fig. 8 C). Together, our findings indicate that TRAPPII resides on a late Golgi/early endosomal compartment that contains Sec7p and Chs3p. Furthermore, this compartment appears to be distinct from PI(3)P-containing endosomal membranes.


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)

TRAPPII largely colocalizes with Sec7p-GFP but not with PI(3)P-containing endosomal membranes. (A, top) Cells containing Sec7p-GFP and Trs120p-13myc were fixed and processed for immunofluorescence. (bottom) Cells containing Trs130p-GFP and Sec7p-DsRed were viewed by direct fluorescence microscopy. (B) Chs3p-GFP–containing puncta also largely colocalized with Sec7p-DsRed. Cells containing Sec7p-DsRed and Chs3p-GFP were viewed by direct fluorescence microscopy. (C) Cells containing Trs130p-GFP and DsRed-FYVE were viewed by direct fluorescence microscopy. Trs130p-GFP did not colocalize with DsRed-FYVE, a marker for PI(3)P-containing endosomal membranes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171297&req=5

fig8: TRAPPII largely colocalizes with Sec7p-GFP but not with PI(3)P-containing endosomal membranes. (A, top) Cells containing Sec7p-GFP and Trs120p-13myc were fixed and processed for immunofluorescence. (bottom) Cells containing Trs130p-GFP and Sec7p-DsRed were viewed by direct fluorescence microscopy. (B) Chs3p-GFP–containing puncta also largely colocalized with Sec7p-DsRed. Cells containing Sec7p-DsRed and Chs3p-GFP were viewed by direct fluorescence microscopy. (C) Cells containing Trs130p-GFP and DsRed-FYVE were viewed by direct fluorescence microscopy. Trs130p-GFP did not colocalize with DsRed-FYVE, a marker for PI(3)P-containing endosomal membranes.
Mentions: Using a monoclonal anti-myc antibody, we performed indirect immunofluorescence to localize Trs120p-myc in cells containing the late Golgi marker Sec7p-GFP. Quantitation of 86 cells revealed that 314 of the 331 Trs120p-myc–containing puncta colocalized with Sec7p-GFP (Fig. 8 A, top). A total of 448 Sec7p-GFP–containing puncta were identified in the 86 cells that were quantitated, and 314 of these puncta colocalized with Trs120p-myc. Similar results were obtained when we examined the localization of Trs130p fused to 3-x-GFP with Sec7p-DsRed (Fig. 8 A, bottom). Furthermore, quantitation of >280 puncta revealed that ∼50% of the Sec7p-DsRed puncta colocalized with Chs3p-GFP and ∼60% of the Chs3p-GFP puncta colocalized with Sec7p-DsRed (Fig. 8 B). Thus, Chs3p and Sec7p largely colocalize with each by direct fluorescence. The localization of TRAPPII was also monitored with respect to the FYVE domain of early endosomal antigen 1, which marks phosphatidylinositol-3-phosphate (PI[3]P)–containing endosomal membranes (Burd and Emr, 1998; Gaullier et al., 1998; Katzmann et al., 2003). Trs130p–3-x-GFP did not colocalize with DsRed-FYVE, which was found on punctate structures adjacent to the vacuole (Fig. 8 C). Together, our findings indicate that TRAPPII resides on a late Golgi/early endosomal compartment that contains Sec7p and Chs3p. Furthermore, this compartment appears to be distinct from PI(3)P-containing endosomal membranes.

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