Fig 5.
Detection of focus-forming metabolic proteins under hypoxia (see also Fig. S4 and Table S3 in the supplemental material). (A) Focus formation by plasmid-introduced recombinant Eno2p. The ΔENO2 strain transformed with various plasmids was semianaerobically cultured at 30°C for 12 h and observed. pYEX-ATG-EGFP, the plasmid used for producing EGFP-FLAG tag; pYEX-ENO2G, the plasmid for producing Eno2p conjugated with the EGFP-FLAG tag; pYEX-ENO2V22AG, the plasmid for producing the Eno2p V22A mutant conjugated with the EGFP-FLAG tag. Bar, 10 μm. Representative data of at least 3 independent experiments are shown. White arrowheads indicate observed foci. (B) Overview of identified proteins coimmunoprecipitated with recombinant Eno2p-EGFP-FLAG and Eno2V22Ap-EGFP-FLAG. Before protein extraction, cells were cultured for 12 h at 30°C semianaerobically. Data represent 2 biological replicates and 2 technical replicates (n = 4). I, 96 proteins coimmunoprecipitated with recombinant Eno2p-EGFP-FLAG and Eno2V22Ap-EGFP-FLAG; II, 29 proteins coimmunoprecipitated with recombinant Eno2p-EGFP-FLAG; III, 16 proteins coimmunoprecipitated with recombinant Eno2V22Ap-EGFP-FLAG. (C) Focus formation by metabolic enzymes and their colocalization with foci formed by N-terminal amino acid sequences conjugated with DsRed. Various GFP strains (of Pfk1p, Fba1p, Tpi1p, Tdh3p, Pgk1p, Gpm1p, and Pyk1p) or strain BY4741 transformed with the plasmid pULR-eno(1-28) were cultured semianaerobically at 30°C for 12 h and observed. Eno(1-28)-DsRed, red fluorescence from the N-terminal (aa 1 to 28) amino acid sequence of Eno2p conjugated with the DsRed monomer (shown in magenta); GFP, green fluorescence from the proteins conjugated with GFP. Bar, 5 μm. White arrowheads indicate colocalized foci (shown in white). (D) Overview of focus-forming proteins in glycolytic pathway under hypoxia. Green or gray characters indicate enzymes that formed or did not form, respectively, foci under hypoxia. (E) Focus formation by metabolic enzymes under hypoxia. Various GFP strains (of Glk1p, Pgi1p, Pfk1p, Fba1p, Tpi1p, Tdh3p, Pgk1p, Gpm1p, Pyk1p, Tal1p, Tkl1p, Pdc1p, Ald6p, Fas1p, Fas2p, and Pyc1p) transformed with the plasmid pULI1 were cultured semianaerobically at 30°C for 12 h (for Pfk1p, Fba1p, Tpi1p, Tdh3p, Pgk1p, Gpm1p, Pyk1p, Tal1p, Tkl1p, Pdc1p, Fas1p, Fas2p, and Pyc1p) or 24 h (for Pgi1p, Glk1p, and Ald6p) and observed. Bar, 10 μm. Green or gray characters indicate enzymes that formed or did not form, respectively, foci under hypoxia.