Figure 5

Glo3p-GFP localization in the presence of over-expressed Sec26p appendage. A. Functionality and localization of Glo3p-GFP. The functionality of the COOH-terminally tagged Glo3p-GFP construct (pRC3341A) was checked by transformation into the heterozygous diploid (MAT a/α his3Δ0/his3Δ0 leu2Δ0/leu2Δ0 ura3Δ0/ura3Δ0 MET15/met15Δ0 LYS2/lys2Δ0 GLO3/glo3Δ::HIS3 GCS1/gcs1Δ:: KAN^R). Following sporulation, haploid strains deleted for the genomic copies of GLO3 and GCS1, were identified. Viable haploids that were His⁺ and KAN^R were also positive for Glo3p-GFP and 5-FOA sensitive. Cells cotransformed with Glo3p-GFP and Sec21p-RFP or Sec7-RFP show coincidence of the green and red fluorescent signal in the punctate pattern typical of the Golgi of S. cerevisiae. B. Glo3p-GFP was expressed in a diploid glo3Δ/glo3Δ gcs1Δgcs1Δ background. This strain was transformed with a plasmid for over-expression of the Sec26p (residues E594-V973; pRC2629b) or Sec21p appendage (residues L676-Q935; pRC2628a) by a copper-inducible promoter (P _CUP1 ). Cells were grown to early log phase, the cultures were divided, and treated with or without 0.7 mM CuSO₄ for 3 hours at room temperature prior to microscopy. C. Selected sec26^tsmutant strains (sec26-1, sec26-2, and sec26^FW) were transformed with the glo3 truncation and point mutation constructs under control of P _GAL1/10 and plated on SCG as a dilution series to evaluate the suppressive ability of the constructs. D. GLO3 suppression of sec26^ts(sec26-1 and sec26-2) requires the ISS motifs located in the COOH-terminus of Glo3p. Mutant sec26 strains were transformed with the indicated constructs and tested for suppression as in 5C. The mutant ISS motif glo3 construct contained alanines at positions 388–390 and 420–422.