Regulation of Rab5 isoforms by transcriptional and post-transcriptional mechanisms in yeast

Abstract

Rab5 GTPases are master regulators of early endosome biogenesis and transport. The genome of Saccharomyces cerevisiae encodes three Rab5 proteins: Vps21, the major isoform, Ypt52 and Ypt53. Here, we show that Vps21 is the most abundant Rab5 protein and Ypt53 is the least abundant. In stressed cells, Ypt53 levels increase but never exceed that of Vps21. Its induction requires the transcription factors Crz1 and Gis1. In growing cells, the expression of Ypt53 is suppressed by post-transcriptional mechanisms mediated by the untranslated regions of the YPT53 mRNA. Based on genetic experiments, these sequences appear to stimulate deadenylation, Pat1-activated decapping and Xrn1-mediated mRNA degradation. Once this regulation is bypassed, Ypt53 protein levels surpass Vps21, and Ypt53 is sufficient to maintain endosomal function and cell growth.

Keywords: Rab5 GTPases; Vps21; Ypt53; mRNA decay; stress.

Figure 1

(A) Protein levels of Rab GTPases in WT and vps4∆ cells (ratio vps4∆/WT) deduced from quantitative proteomics 25. *Significant protein ratio changes 25. n.d., not determined. (B–E) SDS/PAGE and western blot of whole cell protein lysates from logarithmically growing cells analysed with the indicated antibodies. (B) WT, vps4∆ and vps21∆ cells expressing the indicated Rab5 plasmids. Due to strong differences in expression we provide long and short western blot exposures (exp.). For quantifications see Fig. S2B,C,E,F. (C) WT and vps4∆ cells expressing HA‐YPT53 and vps4‐ts or empty plasmid grown into logarithmic phase at permissive temperature (26 °C) and shifted to the restrictive temperature (37 °C) for the indicated time. *Unspecific background bands. For quantification see Fig. S2G. (D) WT cells or the indicated mutants expressing 3xHA‐YPT53. p(recursor) and m(ature) forms of Ape1. For quantification see Fig. S2I. (E) vps21∆, ypt52∆, ypt53∆ cells expressing the indicated plasmids. (F) Growth of vps21∆, ypt52, ypt53∆ cells containing centromeric plasmids expressing 3xHA‐tagged VPS21, YPT52 or YPT53 from their native promoter and terminator sequences or empty vector at the indicated temperatures. (G) Life cell fluorescence microscopy of vps21∆, ypt52∆, ypt53∆ cells expressing Mup1‐GFP (after treatment with 100 μg·mL⁻¹ l‐methionine for 75 min) or GFP‐CPS and the indicated plasmids.

Figure 2

(A) Quantification of YPT53 and VPS21 mRNA (normalized to stable PGK1 mRNA) from logarithmically growing WT (vps4∆ + pRS413‐VPS4) and ESCRT mutant (vps4∆ + pRS413) cells by RT‐qPCR (∆∆C_T method), n = 4. Error bars indicate standard deviation. n.s., not significant. ***P < 0.001. (B) SDS/PAGE and western blot from whole cell protein lysates of WT (BY4742) and congenic crz1∆ and gis1∆ cells in logarithmic phase and after 24 h starvation for amino acids and nitrogen sources analysed with the indicated antibodies. (C) Whole cell protein lysates from logarithmically growing WT (BY4742) cells and the indicated mutants expressing VPS4 or vps4 ^E233Q from plasmids analysed as in (B). (D) Whole cell protein lysates of logarithmically growing WT (BY4742) and indicated congenic deletion strains analysed as in B). *Nonspecific background bands. For quantifications see Fig. S2K,L. (E) Quantification of YPT53 and VPS21 mRNA from logarithmically growing WT (BY4742) and congenic pat1∆ and xrn1∆ cells by RT‐qPCR as in A (n = 3). *P < 0.05, **P < 0.01.

Figure 3

(A) Schematic depicting the chimeric constructs of VPS21 and YPT53 with exchanged upstream and downstream genetic regions. (B) Quantification of YPT53 mRNA normalized to stable PGK1 mRNA from logarithmically growing vps21∆ cells expressing the indicated VPS21/YPT53 chimeric constructs by RT‐qPCR (n ≥ 3). Error bars indicate standard deviation. n.s., not significant, **P < 0.01,*** P < 0.001 (C) SDS/PAGE and western blot of whole cell protein lysates from logarithmically growing vps21∆ or vps4∆ cells expressing the indicated VPS21/YPT53 chimeric constructs and analysed with the indicated antibodies. Due to strong differences in expression we provide long and short western blot exposures (exp.). For quantification see Fig. S2M. (D) Quantification of VPS21 mRNA as in (B) (n = 3), ***P < 0.001. (E) Whole cell protein lysates from logarithmically growing vps21∆ cells expressing the indicated VPS21/YPT53 chimeric constructs and analysed as in (C). p(recursor) and m(ature) forms of Ape1. (F) Whole cell protein lysates of logarithmically growing vps21∆ cells expressing the indicated plasmids treated with 50 μg·mL⁻¹ cycloheximide (CHX) for the indicated times and analysed as in (C). For quantification see Fig. S2N.

Figure 4

(A) Growth of vps21∆, ypt52, ypt53∆ cells expressing the indicated constructs at the indicated temperatures. (B) Life cell fluorescence microscopy of Mup1‐GFP in vps21∆ cells expressing the indicated constructs grown into logarithmic phase and exposed to L‐methionine (100 μg·mL⁻¹) for 90 min. Vac(uoles). Size bars 5 μm. (C) Life cell fluorescence microscopy of vps21∆, ypt52∆, ypt53∆ cells expressing GFP‐CPS and the indicated plasmids. Vac(uoles). Size bars 5 μm. (D) SDS/PAGE and western blot of whole cell protein lysates of logarithmically growing vps21∆ cells expressing the indicated VPS21/YPT53 chimeric constructs and analysed with the indicated antibodies.