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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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Mutants in trs120 and -130 accumulate aberrant membrane structures. Wild-type and mutant cells were shifted to 37°C for 2 h in YPD (yeast extract/peptone/dextrose) medium and processed for EM as described previously (Newman and Ferro-Novick, 1987). (A) Wild type, (B) trs130ts2, (C) trs120-4, and (D) trs120-2. The arrows point to Berkeley bodies or structures that resemble Berkeley bodies. Arrowheads point to vesicles. Bars, 1 μm.
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fig9: Mutants in trs120 and -130 accumulate aberrant membrane structures. Wild-type and mutant cells were shifted to 37°C for 2 h in YPD (yeast extract/peptone/dextrose) medium and processed for EM as described previously (Newman and Ferro-Novick, 1987). (A) Wild type, (B) trs130ts2, (C) trs120-4, and (D) trs120-2. The arrows point to Berkeley bodies or structures that resemble Berkeley bodies. Arrowheads point to vesicles. Bars, 1 μm.

Mentions: Snc1p is the yeast homologue of synaptobrevin. With its homologous partner, Snc2p, it mediates the fusion of post-Golgi vesicles with the plasma membrane (Protopopov et al., 1993). Snc1p largely resides on the plasma membrane but is rapidly endocytosed and traffics through the early endosome before it reaches the late Golgi, where it is incorporated into another round of secretory vesicles (Fig. 4 A; Lewis et al., 2000). To monitor the recycling of GFP-Snc1p, we grew wild-type and mutant cells at the permissive temperature (25°C) or shifted them to 37°C for 60 min. In wild-type cells, GFP-Snc1p was generally found at regions of polarized growth and the plasma membrane. Interestingly, after a 60-min shift to 37°C, little GFP-Snc1p was found at the cell surface in the trs120-2 and -8 mutants (Fig. 4 B). In both mutants, GFP-Snc1p was present on intracellular membranes in >80% of the cells. In trs120-2, GFP-Snc1p was found in small punctate structures at 25 and 37°C. The recycling defect in trs120-2 resulted in a growth defect at 25°C, and when this defect was more pronounced at 37°C, the cells died. An increased cytoplasmic haze was also observed (Fig. 4 B). This cytosolic haze may correspond to the presence of GFP-Snc1p in transport vesicles. In support of this hypothesis, we found that the trs120-2 mutant accumulated three to five times more vesicles than wild type (see Fig. 9). Although no significant defect in invertase secretion was found in the trs120-4 mutant, GFP-Snc1p was found in punctate structures in ∼40% of the cells (Fig. 4 B). Thus, trs120 mutants that exhibited defects in invertase secretion also displayed severe defects in GFP-Snc1p recycling. In contrast, all trs130 mutants were defective in GFP-Snc1p recycling and, like trs120-8, GFP-Snc1p accumulated in larger structures in these mutants (Fig. 4 B, trs130ts2).


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)

Mutants in trs120 and -130 accumulate aberrant membrane structures. Wild-type and mutant cells were shifted to 37°C for 2 h in YPD (yeast extract/peptone/dextrose) medium and processed for EM as described previously (Newman and Ferro-Novick, 1987). (A) Wild type, (B) trs130ts2, (C) trs120-4, and (D) trs120-2. The arrows point to Berkeley bodies or structures that resemble Berkeley bodies. Arrowheads point to vesicles. Bars, 1 μm.
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Related In: Results  -  Collection

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fig9: Mutants in trs120 and -130 accumulate aberrant membrane structures. Wild-type and mutant cells were shifted to 37°C for 2 h in YPD (yeast extract/peptone/dextrose) medium and processed for EM as described previously (Newman and Ferro-Novick, 1987). (A) Wild type, (B) trs130ts2, (C) trs120-4, and (D) trs120-2. The arrows point to Berkeley bodies or structures that resemble Berkeley bodies. Arrowheads point to vesicles. Bars, 1 μm.
Mentions: Snc1p is the yeast homologue of synaptobrevin. With its homologous partner, Snc2p, it mediates the fusion of post-Golgi vesicles with the plasma membrane (Protopopov et al., 1993). Snc1p largely resides on the plasma membrane but is rapidly endocytosed and traffics through the early endosome before it reaches the late Golgi, where it is incorporated into another round of secretory vesicles (Fig. 4 A; Lewis et al., 2000). To monitor the recycling of GFP-Snc1p, we grew wild-type and mutant cells at the permissive temperature (25°C) or shifted them to 37°C for 60 min. In wild-type cells, GFP-Snc1p was generally found at regions of polarized growth and the plasma membrane. Interestingly, after a 60-min shift to 37°C, little GFP-Snc1p was found at the cell surface in the trs120-2 and -8 mutants (Fig. 4 B). In both mutants, GFP-Snc1p was present on intracellular membranes in >80% of the cells. In trs120-2, GFP-Snc1p was found in small punctate structures at 25 and 37°C. The recycling defect in trs120-2 resulted in a growth defect at 25°C, and when this defect was more pronounced at 37°C, the cells died. An increased cytoplasmic haze was also observed (Fig. 4 B). This cytosolic haze may correspond to the presence of GFP-Snc1p in transport vesicles. In support of this hypothesis, we found that the trs120-2 mutant accumulated three to five times more vesicles than wild type (see Fig. 9). Although no significant defect in invertase secretion was found in the trs120-4 mutant, GFP-Snc1p was found in punctate structures in ∼40% of the cells (Fig. 4 B). Thus, trs120 mutants that exhibited defects in invertase secretion also displayed severe defects in GFP-Snc1p recycling. In contrast, all trs130 mutants were defective in GFP-Snc1p recycling and, like trs120-8, GFP-Snc1p accumulated in larger structures in these mutants (Fig. 4 B, trs130ts2).

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