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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 analysis of CPY trafficking in trs130 and -120 mutants. (A) Mutants in trs130, but not -120, display a defect in CPY trafficking. Wild-type (WT) and mutant cells were grown at 25°C, preincubated at 37°C for 20 min, labeled for 4 min, and chased for 30 min. Aliquots of samples were removed at the indicated time points and immunoprecipitated with anti-CPY antibody. (B) Mutants in trs130 and -120 do not secrete CPY into the growth medium. The growth medium was collected from wild-type and mutant cells that were shifted to 37°C for 2 h, processed for TCA precipitation, and blotted with anti-CPY antibody. The secretion of CPY from the vps1Δ mutant is shown as a control.
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fig2: The analysis of CPY trafficking in trs130 and -120 mutants. (A) Mutants in trs130, but not -120, display a defect in CPY trafficking. Wild-type (WT) and mutant cells were grown at 25°C, preincubated at 37°C for 20 min, labeled for 4 min, and chased for 30 min. Aliquots of samples were removed at the indicated time points and immunoprecipitated with anti-CPY antibody. (B) Mutants in trs130 and -120 do not secrete CPY into the growth medium. The growth medium was collected from wild-type and mutant cells that were shifted to 37°C for 2 h, processed for TCA precipitation, and blotted with anti-CPY antibody. The secretion of CPY from the vps1Δ mutant is shown as a control.

Mentions: To monitor the trafficking of a second marker protein, we analyzed the vacuolar protease CPY. CPY is synthesized, translocated, and glycosylated in the lumen of the ER (p1 form) before it is transported to the Golgi (p2), where it receives additional carbohydrate modifications. On transport to the vacuole, CPY is proteolytically activated to the mature form (Stevens et al., 1982). To examine CPY trafficking, wild-type and mutant cells were preincubated at 37°C for 20 min, pulse labeled for 4 min, and chased for 30 min (Fig. 2 A). At the end of the chase, all trs130 mutants accumulated small amounts of the p1 form of CPY as well as a heterogeneous smear of CPY that accumulated between the p1 and p2 forms of CPY. Small amounts of mature CPY were also observed at the end of the chase (Fig. 2 A, top), suggesting that Trs130p is not completely inactivated at 37°C. These results are consistent with our previous proposal that TRAPPII may be required for intra-Golgi traffic (Sacher et al., 2001). This finding is also consistent with the observation that fully glycosylated invertase accumulates in the late Golgi in all trs130 mutants. In contrast, the kinetics of anterograde CPY trafficking in all trs120 mutants was the same as wild type (Fig. 2 A, bottom).


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 analysis of CPY trafficking in trs130 and -120 mutants. (A) Mutants in trs130, but not -120, display a defect in CPY trafficking. Wild-type (WT) and mutant cells were grown at 25°C, preincubated at 37°C for 20 min, labeled for 4 min, and chased for 30 min. Aliquots of samples were removed at the indicated time points and immunoprecipitated with anti-CPY antibody. (B) Mutants in trs130 and -120 do not secrete CPY into the growth medium. The growth medium was collected from wild-type and mutant cells that were shifted to 37°C for 2 h, processed for TCA precipitation, and blotted with anti-CPY antibody. The secretion of CPY from the vps1Δ mutant is shown as a control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171297&req=5

fig2: The analysis of CPY trafficking in trs130 and -120 mutants. (A) Mutants in trs130, but not -120, display a defect in CPY trafficking. Wild-type (WT) and mutant cells were grown at 25°C, preincubated at 37°C for 20 min, labeled for 4 min, and chased for 30 min. Aliquots of samples were removed at the indicated time points and immunoprecipitated with anti-CPY antibody. (B) Mutants in trs130 and -120 do not secrete CPY into the growth medium. The growth medium was collected from wild-type and mutant cells that were shifted to 37°C for 2 h, processed for TCA precipitation, and blotted with anti-CPY antibody. The secretion of CPY from the vps1Δ mutant is shown as a control.
Mentions: To monitor the trafficking of a second marker protein, we analyzed the vacuolar protease CPY. CPY is synthesized, translocated, and glycosylated in the lumen of the ER (p1 form) before it is transported to the Golgi (p2), where it receives additional carbohydrate modifications. On transport to the vacuole, CPY is proteolytically activated to the mature form (Stevens et al., 1982). To examine CPY trafficking, wild-type and mutant cells were preincubated at 37°C for 20 min, pulse labeled for 4 min, and chased for 30 min (Fig. 2 A). At the end of the chase, all trs130 mutants accumulated small amounts of the p1 form of CPY as well as a heterogeneous smear of CPY that accumulated between the p1 and p2 forms of CPY. Small amounts of mature CPY were also observed at the end of the chase (Fig. 2 A, top), suggesting that Trs130p is not completely inactivated at 37°C. These results are consistent with our previous proposal that TRAPPII may be required for intra-Golgi traffic (Sacher et al., 2001). This finding is also consistent with the observation that fully glycosylated invertase accumulates in the late Golgi in all trs130 mutants. In contrast, the kinetics of anterograde CPY trafficking in all trs120 mutants was the same as wild type (Fig. 2 A, bottom).

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