JCS042457 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. The multicopy SFT2 vector carries a complementing copy of SFT2 that cannot suppress the growth phenotypes of yip1- or yif1- thermosensitive strains. (A) CBY1769 was transformed with 2µ SFT2 URA3, sporulated and tetrads were dissected and scored. Here, four independent haploid strains for this dissection with the indicated genotype and harboring 2µ SFT2 were grown for 48 hours at 30°C in the presence (YMD-URA) or absence (5-FOA) of the SFT2 plasmid. This result confirms the got1Δ sft2Δ synthetic lethal interaction reported previously (Conchon et al., 1999), and proves that the SFT2 plasmid can rescue this phenotype. The positive control strain CBY901 (WT 2µ SLY1-20 URA3) grew on 5-FOA, whereas the negative control strain CBY903 (ypt1Δ::HIS3 2µ SLY1-20 URA3) did not. (B) WT (FY834) and yif1-2 (YHM60) were transformed with 2µ SFT2. Two transformants per strains were grown on YPD for 48 hours at permissive (25°C) and restrictive (37°C) temperatures. As described in the text and in Table 1, 2µ GOT1 partially rescued the growth defect of the yif1-2 strain, while 2µ YIF1 fully rescued its defect to wild-type levels. However, 2µ SFT2 was unable to rescue the yif1-2 growth defect. We obtained similar results for the suppression of the growth defects of yif1-4, yip1-1, yip1-2 and yip1-4 (data not shown).

• Supplemental Figure S2 -

  Fig. S2. Multicopy GOT1 suppresses a subset of thermosensitive SEC mutants. Strains with temperature-sensitive mutations in genes required for early secretory pathway function were transformed with empty 2µ vector (vec) or multicopy GOT1 (GOT1). Two independent transformants were grown to saturation in appropriate medium to maintain plasmid selection, adjusted to an OD(600) of 1.0 and 3 µl of tenfold dilutions were spotted onto YPD plates. Plates were incubated at 30°C, 34°C or 38°C for 48 hours. A representative set of strains is shown in this figure and a summary of all strains tested is shown in Table 1.

• Supplemental Figure S3 -

  Fig. S3. Multicopy GOT1 does not suppress a yip1Δ deletion mutation. (A) CBY1769 was transformed with 2µ GOT1 URA3, sporulated and tetrads were dissected and scored as described in the legend for supplementary material Fig. S1. This result confirms the got1Δ sft2Δ synthetic lethal interaction reported previously (Conchon et al., 1999), and demonstrates the GOT1 plasmid complements. (B) CBY1319 (yip1Δ::KAN^R CEN YIP1 URA3) was transformed with 2µ LEU2 or 2µ GOT1 LEU2. Transformants were grown in parallel in YMD-LEU-URA or 5-FOA plates and incubated for 48 hours at 25°C. CBY1319, which contains a yip1Δ deletion mutation and is complemented by a YIP1-URA3 plasmid, cannot grow on 5-FOA even in the presence of multi-copy GOT1. The positive control strain CBY901 (WT 2µ SLY1-20 URA3) transformed with 2µ LEU2 grew on 5-FOA whereas the negative control strain CBY903 (ypt1Δ::HIS3 2µ SLY1-20 URA3) transformed with 2µ LEU2 did not.

• Supplemental Figure S4 -

  Fig. S4. The original got1Δ strain displays altered expression and distribution of Ypt proteins. Semi-intact cells from wild-type and got1Δ cells transformed with empty 2µ vector or 2µ GOT1 were separated into soluble and membrane-bound fractions by differential centrifugation. Proteins in the total (T), soluble fraction (S) and membrane bound pellet (P) were detected by immunoblot. (A) The got1Δ membranes prepared from SCY02 (Conchon et al., 1999) contain increased levels of soluble Ypt1p and Ypt7p compared to the isogenic wild type (SEY6210). This alteration was not corrected by transformation with 2µ GOT1. (B) Wild-type (CBY740) and got1Δ (CBY1574) membranes from the Research Genetics collection do not display altered Ypt protein expression patterns.

• Supplemental Figure S5 -

  Fig. S5. Quantification of ER and Golgi morphologies in wild type and GAL1-3HA-GOT1 cells. Cell cultures as described in Figure 8 were shifted to 2% galactose and inspected by microscopy. (A) WT (CBY2537) and GAL1-3HA-GOT1 (CBY2542) cells expressing Sec63-GFP were scored for the appearance of elaborated and gapped ER structures in focal planes near the center of a cell. 20% of wild type cells (n=233) exhibited such structures whereas 60% of the GAL1-3HA-GOT1 cells (n=189) abnormal ER structures. (B) WT (CBY2538) and GAL1-3HA-GOT1 (CBY2543) cells expressing Sec7-GFP were scored for a diffuse fluorescence pattern and loss of punctate structures. Only 20% of wild type cells (n=237) exhibited altered Sec7-GFP fluorescence whereas 95% of the GAL1-3HA-GOT1 cells (n=302) displayed diffuse Sec7-GFP fluorescence with or without loss of Sec7-GFP puncta.