A Rab requirement is not bypassed in SLY1-20 suppression

Abstract

The Rab GTPase Ypt1p and the large homodimer Uso1p are both required for tethering endoplasmic reticulum-derived vesicles to early Golgi compartments in yeast. Loss-of-function ypt1 and uso1 mutations are suppressed by SLY1-20, a dominant allele that encodes the Sed5p-associated protein, Sly1p. Here, we investigate the mechanism of SLY1-20 suppression. In wild-type strains, Ypt1p can be coimmunoprecipitated with Uso1p; however, in a ypt1Delta/SLY1-20 strain, which lacks this complex, membrane binding of Uso1p was reduced. In spite of Ypt1p depletion, Uso1p-dependent vesicle tethering was not bypassed under the ypt1Delta/SLY1-20 condition. Moreover, tethering and fusion assays with ypt1Delta/SLY1-20 membranes remained sensitive to Rab GDP dissociation inhibitor. These results indicate that an alternative Rab protein satisfies the Ypt1p requirement in Uso1p-dependent tethering when SLY1-20 is expressed. Further genetic and biochemical tests revealed that a related Rab protein, Ypt6, might substitute for Ypt1p in ypt1Delta/SLY1-20 cells. Additional experimentation to address the mechanism of SLY1-20 suppression in a cog2Delta [sec35Delta] strain indicated that the Cog2p subunit of the conserved oligomeric Golgi complex is either functionally redundant or is not directly required for anterograde transport to the Golgi complex.

Figure 1.

Ypt1p function influences Uso1p membrane association. (A) WT (CBY1550) and ypt1-3 (CBY474) semi-intact cells were incubated at 29°C with ATP/GTP in buffer for 10 min. High-speed supernatant and membrane pellet fractions were isolated by centrifugation at 100,000 × g. The total (T), supernatant (S), and resuspended pellet (P) fractions were diluted into SDS-PAGE sample buffer and analyzed by Western blot. (B) The experiment is similar to that in A, except it uses semi-intact cells from WT (CBY900), wild-type with pSLY1-20 (CBY901), and ypt1Δ/SLY1-20 (CBY903) strains.

Figure 2.

Ypt1p coimmunoprecipitates with Uso1p. (A) Semi-intact cells from WT (CBY1550) and WT with 2 μm pUso1p-myc (CBY528) were washed in buffer to remove cytosol. Washed membranes were incubated with or without ATP/GTP and GDI at 20°C for 30 min, followed by solubilization in 1% digitonin at 25°C for 12 min. Solubilized membranes were immunoprecipitated with anti-myc monoclonal antibodies and protein A beads at 4°C for 1 h. Washed beads were resuspended in SDS-PAGE sample buffer and analyzed by Western blot. In this experiment, the total lanes (T) represent 0.6% of the total immunoprecipitated (IP) input. (B) The experiment is similar to that in A, except immunoprecipitation was carried out using semi-intact cells from a strain with endogenously HA-tagged USO1 (CBY1189) and an isogenic wild-type (CBY1187). Anti-HA precipitation was performed using HA monoclonal antibodies. The T lanes represent 2.0% of the total IP input.

Figure 3.

In vitro transport, budding, and tethering in the absence of Ypt1p. (A) Transport kinetics was measured in WT (CBY901) and ypt1Δ/SLY1-20 (CBY903) semi-intact cells in the presence of purified transport factors (COPII, Uso1p, and LMA1) at 0, 15, 30, 45, and 60 min. The percentage of transport represents the amount of [³⁵S]gp-α-factor that has been modified by the addition of Golgi-specific α1,6-mannose residues. (B) Budding and tethering in WT (CBY901) and ypt1Δ/SLY1-20 (CBY903) semi-intact cells was assayed with COPII and Uso1p proteins at 23°C for 30 min. The percentage of diffusible vesicles represents the amount of [³⁵S]gp-α-factor released into a medium-speed supernatant fraction divided by the total amount of [³⁵S]gp-α-factor contained in the reaction.

Figure 4.

GDI inhibits transport in the absence of Ypt1p. Transport in WT (CBY901) and ypt1Δ/SLY1-20 (CBY903) semi-intact cells in the presence of purified transport factors (COPII, Uso1p, and LMA1) and increasing amounts of GDI (micromolar) at 23°C for 70 min. The percentage of transport represents the amount of [³⁵S]gp-α-factor that has been modified by the addition of Golgi-specific α-1,6-mannose residues.

Figure 5.

Transport and tethering in the absence of Ypt1p requires Uso1p and is sensitive to GDI. (A) COPII vesicles were isolated in vitro from wild-type membranes at 20°C and incubated in a second transport stage with WT (CBY901) or ypt1Δ/SLY1-20 (CBY903) acceptor membranes with buffer alone, indicated transport factors and transport factors plus GDI at 23°C for 60 min. The percentage of transport represents the amount of [³⁵S]gp-α-factor that has been modified by the addition of Golgi-specific α-1,6-mannose residues. (B) COPII vesicles were isolated as described in A and were incubated in a second stage tethering reaction with WT (CBY901) or ypt1Δ/SLY1-20 (CBY903) acceptor membranes in the presence of buffer, Uso1p, or Uso1p plus GDI. The percentage of diffusible vesicles represents the amount of [³⁵S]gp-α-factor released into a medium-speed supernatant fraction divided by the total amount of [³⁵S]gp-α-factor contained in the reaction.

Figure 6.

SLY1-20 does not suppress a double ypt1Δ uso1Δ strain. Serial dilutions of WT/pSLY1-20 (CBY901), uso1Δ/pUSO1 pSLY1-20 (CBY1381), uso1Δ/pUSO1 (CBY1297), and ypt1Δ uso1Δ/pUSO1 pSLY1-20 (CBY1362) strains were spotted on YPD (A) or minimal 5-FOA (B) plates and incubated at 24°C.

Figure 7.

SLY1-20 does not suppress ypt1Δ ypt6Δ. Serial dilutions of uso1Δ/pUSO1 pSLY1-20 (CBY1381), ypt1Δ/pSLY1-20 (CBY903), ypt1Δ ypt31Δ/pYPT1 pSLY1-20 (CBY1548), ypt1Δ ypt32Δ/pYPT1 pSLY1-20 (CBY1399), and ypt1Δ ypt6Δ/pYPT1 pSLY1-20 (CBY1396) were spotted on YPD (A) or minimal 5-FOA (B) plates and incubated at 24°C.

Figure 8.

Ypt6p is up-regulated in ypt1Δ/SLY1-20 cells. Equal protein amounts of semi-intact cells from WT (CBY740), ypt6Δ (CBY1344), WT/SLY1-20 (CBY901), ypt1Δ/SLY1-20 (CBY903), and ypt32Δ (CBY1343) strains were washed once with buffer 88 and resuspended in equal amounts of buffer 88 and SDS PAGE sample buffer. For Western analysis, protein samples were separated on 12.5% gels and blotted with anti-Ypt1p, anti-Ypt6p, anti-Erv41p, and anti-Ypt7p polyclonal antibodies.

Figure 9.

Loss of Cog2p does not influence the association of known Golgi-tethering factors. WT/SLY1-20 (CBY1020) and cog2Δ/SLY1-20 (CBY1021) semi-intact cells were incubated at 29°C with ATP/GTP in buffer 88 for 10 min. High-speed supernatant and -membrane pellet fractions were isolated by centrifugation at 100,000 × g. The total, supernatant, and resuspended pellet fractions were diluted in SDS-PAGE sample buffer and analyzed by Western blot.

Figure 10.

Transport, budding, and tethering in the absence of Cog2p (A) Transport in semi-intact cells from WT/SLY1-20 (CBY1020) and cog2Δ/SLY1-20 (CBY1021) was assayed with buffer 88 alone or with purified reconstitution factors (COPII, Uso1p, and LMA1) at 23°C for 70 min. The percentage of transport represents the amount of [³⁵S]gp-α-factor that has been modified by the addition of Golgi-specific α1,6-mannose residues. (B) Budding and tethering in the same semi-intact cells was assayed with COPII and Uso1p proteins at 23°C for 30 min. The percentage of diffusible vesicles represents the amount of [³⁵S]gp-α-factor released into a medium-speed supernatant fraction divided by the total amount of [³⁵S]gp-α-factor contained in the reaction.