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

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.

Figure 1.

The analysis of invertase secretion in trs130 and -120 mutants. (A) A schematic diagram of trs120 and -130 mutants used in this study. The trs120-1 mutant was made by truncating 481 amino acids from the COOH terminus of Trs120p. The other trs120 mutants were made by transposon mutagenesis as described in Materials and methods. The arrows point to the position where the transposon is inserted in each mutant. Five trs130 mutants were made by truncating 33, 54, 74, 94, and 124 amino acids from the COOH terminus of Trs130p. Invertase secretion is blocked in all trs130 mutants (B) but not all trs120 mutants (C). Wild-type (WT) and mutant cells were grown at 25°C and preincubated at 37°C for 20 min. Cells were resuspended in low-glucose medium to induce the synthesis of invertase and were radiolabeled for 60 min. Samples were then processed for the immunoprecipitation of internal (I) and periplasmic (E) invertase. To facilitate the detection of the high molecular mass form of invertase in trs130 mutants, cells were transformed with the SUC2 gene on a 2-μm plasmid.

Figure 2.

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.

Figure 3.

Mutants in trs120 do not block general secretion. Cells were grown to early log phase at 25°C, preincubated to 37°C for 20 min, radiolabeled for 15 min, and then chased for 30 min. The growth medium was separated from the cells by centrifugation, processed for TCA precipitation, and analyzed by SDS-PAGE on a 10% gel as described previously (Kim et al., 2001). The asterisks mark proteins secreted into the medium in wild-type (WT) cells.

Figure 4.

Mutants in trs120 and - 130 are defective in GFP-Snc1p recycling. (A) The recycling pathway of GFP-Snc1p. (B) GFP-Snc1p was examined in wild-type (WT) and mutant cells that were grown at 25°C (0 min) or shifted to 37°C for 60 min. (C) GFP-Snc1p was examined in wild type, trs130^ts2, and trs120-8 mutants at 0, 5, 15, 30, and 60 min after a shift to 37°C.

Figure 5.

GFP-Snc1p localizes to early endosomes in the trs130^ts2 and - 120-8 mutants. Cells were labeled with FM4-64 as described in Materials and methods. FM4-64 was internalized for 5 or 30 min.

Figure 6.

Mutants in trs120 and - 130 are defective in the trafficking of Chs3p-GFP. Chs3p-GFP was examined in wild-type and mutant cells that were grown at 25°C or shifted to 37°C for 30 min. Arrows point to bud necks and incipient bud sites, which are visible in the differential interference contrast (DIC) image.

Figure 7.

Trs120p resides on a late Golgi/early endosomal compartment. Lysates were prepared and fractionated on a sucrose density gradient as described in Materials and methods. Trs120p does not colocalize with the early Golgi marker Och1p (A) or the ER-Golgi SNARE Sec22p (B). Trs120p largely cofractionates with the late Golgi/early endosomal marker Chs3p (C).

Figure 8.

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.

Figure 9.

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) trs130 ^ts2, (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.

Figure 10.

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.