Establishing a role for the GTPase Ypt1p at the late Golgi

Abstract

GTPases of the Rab family cycle between an inactive (GDP-bound) and active (GTP-bound) conformation. The active form of the Rab regulates a variety of cellular functions via multiple effectors. Guanine nucleotide exchange factors (GEFs) activate Rabs by accelerating the exchange of GDP for GTP, while GTPase activating proteins (GAPs) inactivate Rabs by stimulating the hydrolysis of GTP. The GTPase Ypt1p is required for endoplasmic reticulum (ER)-Golgi and intra-Golgi traffic in the yeast Saccharomyces cerevisiae. Recent findings, however, have shown that Ypt1p GEF, GAP and an effector are all required for traffic from the early endosome to the Golgi. Here we describe a screen for ypt1 mutants that block traffic from the early endosome to the late Golgi, but not general secretion. This screen has led to the identification of a collection of recessive and dominant mutants that block traffic from the early endosome. While it has long been known that Ypt1p regulates the flow of biosynthetic traffic into the cis side of the Golgi, these findings have established a role for Ypt1p in the regulation of early endosome-Golgi traffic. We propose that Ypt1p regulates the flow of traffic into the cis and trans side of the Golgi via multiple effectors.

Keywords: Golgi; Rab; early endosome; membrane traffic.

Figure 1

Summary of ypt1 mutant screen. (A) The Table shows the results from the mutant screen. (B) Wild type, ypt1, and gyp1Δ strains were spotted on YPD plates, incubated with a nitrocellulose filter and blotted for secreted CPY (left panel) and the cytosolic protein Adh1p (right panel), which was used as a lysis control.

Figure 2

The ypt1 mutant alleles display GFP-Snc1p recycling defects. GFP-Snc1p was examined in wild type, the ypt1 mutants and the gyp1Δ deletion strain. All strains were grown in selective media at 25°C.

Figure 3

GFP-Snc1p accumulates in early endosomes in the ypt1-53 mutant. Wild type and the ypt1-53 mutant, expressing GFP-Snc1p, were incubated with FM4-64 for 5 min or 60 min to label early endosomes and the vacuole, respectively. Arrows point to GFP-Snc1p structures that label with FM4-64 (bottom left).

Figure 4

Analysis of CPY trafficking and general secretion in the ypt1 mutants. (A) Lysates were prepared from wild type and the ypt1 mutants grown at 25°C (top) or shifted to 37°C for 2 hr (bottom) and then blotted with anti-CPY and anti-Bos1p antibodies. Bos1p was used as a loading control. The sec18-1 mutant, shifted to 37°C for 2 hr, was used to identify p1CPY. (B) CPY was processed normally in the ypt1 mutants at 37°C. Wild type, ypt1, and sec18-1 strains grown at 25°C were shifted to 37°C for 20 min, pulse labeled for 4 min, and then chased. Samples were taken before (0 min) and after the chase (30 min). CPY was immunoprecipitated and analyzed for processing defects on an 8% SDS polyacrylamide gel. (C) General secretion is normal in the ypt1-53 and ypt1-151 mutants. Wild type, ypt1, and sec18-1 strains were pulse labeled for 5 min and then chased for 20 min. Proteins secreted into the medium were TCA precipitated and resolved on an 8% SDS polyacrylamide gel.

Figure 5

The ypt1 mutants exhibit FM4-64 recycling defects. Cells were allowed to internalize FM4-64 for 12 min at 25°C and then washed thoroughly with ice cold media. Fluorescence was measured every second for 10 min (600 sec total) immediately following resuspension in 25°C media. Data is plotted as % fluorescence over time. FM4-64 recycling in the gyp1Δ (A), ypt1-52, ypt1-151 (B), ypt1-53, ypt1-132 (C), ypt1-138, and ypt1-166 (D) mutants was compared to wild type (A,B,C, D). All strains were tested a minimum of three times and the data from one representative experiment is shown.

Figure 6

Ypt1p colocalizes with the late Golgi marker Sec7p. Cells containing mCherry-Ypt1p (mCh-Ypt1p) and Sec7-GFP (NY2804) were analyzed with a spinning disc confocal microscope (Top panel). Arrows (Top panel, right) point to compartments that display colocalization. The graph (bottom panel) shows the percent of mCh-Ypt1p puncta that colocalize with Sec7-GFP puncta. Scale bar = 5μm, error bars = s.d.

Figure 7

The ypt1-53 mutant accumulates clusters of vesicles. The trs130-2 (B) and ypt1-3 (C) mutants were grown in YPD medium at 25°C and shifted to 37°C for 2 hr prior to preparation for EM analysis. The EM phenotype of wild type was the same with cells grown at 25°C (A) or shifted to 37°C (not shown). The vacuole is fragmented in the ypt1-53 mutant and roughly 25% of the cells contained a cluster of 60-70 nm vesicles (D, E, see arrow). The inset in panel D is magnified and shown in E. All size bars are 1 μm. Abbreviations are as follows: vacuole (Vac), Endoplasmic Reticulum (ER), vesicles (V), Berkeley bodies (B).

Figure 8

A summary of mutations in the new ypt1 mutants All mutated amino acid changes are shown in (A) and a sequence alignment is shown in (B). The switch I and switch II effector regions (11) are highlighted or underlined (A, B). Mutations in bold were introduced individually or in combination by site-directed mutagenesis (A, B), dots indicate no mutation (B). (C) A ribbon diagram of Ypt1p in its active form (PDB 1D 1YZN). GPPNP (GTP analog) and Mg²⁺ in the active site are lavender. Residues that were mutated are shown in red (Q176, N177 and N179 at the very C-terminus are not in the structure and are not shown).

Figure 9

Single mutations in ypt1 can recapitulate the GFP-Snc1p recycling defect seen in the ypt1-53 and ypt1-132 mutants. (A) The recycling defect in the ypt1-53 mutant appears to be the consequence of the I41M mutation. Images of wild type, ypt1-53, ypt1^I41M, ypt1^K46E, and ypt1^Q176L expressing GFP-Snc1p cells are shown. (B) The recycling defect in the ypt1-132 mutant appears to be the consequence of the T40A mutation. Images of the ypt1-132 and ypt1^T40A mutants expressing GFP-Snc1p are shown.

Figure 10

The ypt1-19 mutant is dominant. The mutations in the ypt1-19 mutant were analyzed for recycling defects. Only the ypt1^F162V and ypt1^D6V,F162V mutations were defective in GFP-Snc1p recycling. The dominant recycling defect in the ypt1-19 mutant appears to be the consequence of the ypt1^D6V,F162V mutations.