A Rab GAP cascade defines the boundary between two Rab GTPases on the secretory pathway

Abstract

Membrane traffic along the endocytic and exocytic pathways relies on the appropriate localization and activation of a series of different Rab GTPases. Rabs are activated by specific guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). GEF cascades, in which one Rab in its GTP-bound form recruits the GEF that activates the next Rab along the pathway, can account for the sequential activation of a series of Rabs, but it does not explain how the first Rab is inactivated after the next Rab has been activated. We present evidence for a counter-current GAP cascade that serves to restrict the spatial and temporal overlap of 2 Rabs, Ypt1p and Ypt32p, on the exocytic pathway in Saccharomyces cerevisiae. We show that Gyp1p, a GAP for Ypt1p, specifically interacts with Ypt32p, and that this interaction is important for the localization and stability of Gyp1p. Moreover, we demonstrate that, in WT cells, Ypt1p compartments are converted over time into Ypt32p compartments, whereas in gyp1Delta cells there is a significant increase in compartments containing both proteins that reflects a slower transition from Ypt1p to Ypt32p. GEF cascades working in concert with counter-current GAP cascades could generate a programmed series of Rab conversions responsible for regulating the choreography of membrane traffic.

Fig. 1.

Gyp1p specifically interacts with active Ypt32p. (A) A yeast 2-hybrid assay between GYP1 and YPT32, YPT1 or the hydrolysis-deficient YPT32-Q72L, and YPT1-Q67L alleles. Growth on solid medium lacking leucine/tryptophan (LW) indicates the presence of the plasmids, growth on solid medium lacking leucine/tryptophan/histidine + 10 mM 3-AT (3-AT) indicates a positive 2-hybrid interaction (boxed panel). (B) Yeast 2-hybrid analysis between GYP1 and YPT32 mutant alleles that mimic GDP- (YPT32-S27N) or GTP- (YPT32-Q72L) bound states. Growth on solid medium lacking leucine/tryptophan/histidine + 10 mM 3-AT medium indicates a positive 2-hybrid interaction. (C) Diagrams of the GST-Gyp1 construct that were used for the in vitro binding experiment in D. The GAP catalytic domain (TBC) of Gyp1p is shown. (D) Lysates of yeast strains over-expressing HA-Ypt32p, HA-Ypt1p, or HA-Sec4p were incubated with the various GST-Gyp1 fusion proteins shown in C. Lanes 1 and 2 represent duplicates of the binding reactions. Binding was detected by Western blot using antibodies specific to each GTPase. A 2% input of yeast total lysate was run for each GTPase. Ponceau S staining of one of the membranes shows the presence of the various GST-Gyps constructs. (E) Western blot against HA to detect the level of expression of each HA-Ypts on the lysates used for the binding experiments.

Fig. 2.

Gyp1p levels and localization depend on its interaction with functional Ypt32p. (A) GFP-Gyp1p localization in WT (NY2772) and ypt32ts (NY2773) cells at 25 °C. (B) GFP-HA-Gyp1p levels in total protein lysates from WT or ypt32ts (2 independent samples) detected by immunoblotting for the HA epitope. A Ypt1p immunoblot is shown as loading control. (C) Immunoblotting for HA to analyze the levels of mCherry-HA-Gyp1p (CH-HA-Gyp1) in WT (NY2775) cells, ypt32ts (NY2776) cells, or ypt32ts cells expressing functional GFP-YPT32 (NY2777). (D) A Gyp1p construct lacking the Ypt32p-interaction region (GFP-C-Gyp1p; NY2774) is mis-localized in comparison to the localization of full-length Gyp1p (GFP-Gyp1p) in WT cells. (Scale bar, 5 μm in A and D.) (E) Subcellular fractionation of GFP-Gyp1p and GFP-C-Gyp1p. TL lane represents 2% of the total protein lysate that was fractionated. P13 and P100 represent the membrane-bound fractions obtained after 10,000 × g and 100,000 × g centrifugations, respectively. S100 represents the soluble fraction obtained after the 100,000 × g centrifugation. An equal amount of sample was loaded for each fraction. Immunoblotting for GFP was used to detect GFP-Gyp1p or GFP-C-Gyp1p. Immunoblots for Gos1p (membrane) and Adh1p (cytosolic) are shown as controls. (F) Quantification of the data shown in E. Image acquisition data are provided in SI Methods.

Fig. 3.

Co-localization of Gyp1p with Ypt32p and the role of Gyp1p in limiting co-localization of Ypt1p and Ypt32p. (A) Fluorescent images of gyp1 cells expressing GFP-Ytp32p and CH-Gyp1p (NY2778). Merged fluorescent images were superimposed with the bright-field image (Lower Right) to show the cell contour. Arrows indicate spots where GFP-Ypt32p and CH-Gyp1p co-localized (yellow spots). (B) Fluorescent images from gyp1 cells expressing GFP-Gyp1p and CH-Ypt1p (NY2779). Arrow indicates a spot with co-localized GFP-Gyp1p and CH-Ypt1p. (C) Bar graph shows the percentage of co-localization of the following proteins: GFP-Ypt32p spots containing CH-Gyp1p signal (cyan bar), CH-Gyp1p spots containing GFP-Ypt32p signal (magenta bar), GFP-Gyp1p spots containing CH-Ypt1p (white bar), and CH-Ypt1p spots containing GFP-Gyp1p (gray bar; ≈100 cells, 2–5 spots/cell, error bar indicates SD); **P < 0.001 (t test) between Gyp1p/Ypt32p and Gyp1p/Ypt1p. (D) Fluorescent images of WT cells expressing GFP-Ypt32p and CH-Ypt1p (NY2780). Arrow indicates a spot with both GFP-Ypt32p and CH-Ypt1p. (E) Fluorescent images of gyp1 cells expressing GFP-Ytp32p and CH-Ypt1p (NY2781). Arrows indicate spots in which GFP-Ypt32p and CH-Gyp1p co-localize. (F) Bar graph shows the percentage of compartments in which GFP-Ypt32p and CH-Ypt1p (cyan bars) co-localize, or compartments in which CH-Ypt1p and GFP-Ypt32p (magenta bars) co-localize in WT and gyp1 cells (≈100 cells, 2–5 spots/cell, error bar indicates SD); **P < 0.001 (t test) between WT and gyp1. (Scale bar, 5 μm.) Image acquisition data are provided in SI Methods.

Fig. 4.

Three-dimensional time-lapse fluorescence microscopy demonstrates the conversion of a Ypt1p compartment to a Ypt32p compartment. (A) Images from Movie S1 demonstrate conversion of a CH-Ypt1p compartment to a GFP-Ypt32p compartment (arrows) in WT cells (NY2780). Numbers indicate the time in seconds relative to the frame where the percent of total signal was 50% (i.e., time 0). White spots represent the co-localization between CH-Ypt1p and GFP-Ypt32p. (B) Averaged trace of CH-Ypt1p and GFP-Ypt32p signals in WT cells (n = 16, error bars indicate SD); **P < 0.001 (t test) between Ypt1p and Ypt32p signals. (C) Images from Movie S3 demonstrate conversion of a CH-Ypt1p compartment to a GFP-Ypt32p compartment (arrows) in gyp1 cells (NY2781). (D) Averaged trace of CH-Ypt1p and GFP-Ypt32p signals in gyp1 cells (n = 18, error bars indicate SD); *P < 0.007 (t test) between Ypt1p and Ypt32p signals. (Scale bar, 5 μm.) Image acquisition data are provided in SI Methods.