Figure 1.
Growth phenotypes of pUG-trs20 and bet3-11 mutants.
Serial dilutions were spotted onto YPD plates and incubated for 3 days at the indicated temperatures. (A) Temperature-sensitive phenotype of Trs20 mutants (Δtrs20 cells containing plasmid pUG23-trs20-1 or pUG23-trs20-6) compared to cells expressing a WT copy of Trs20 (Δtrs20 cells containing plasmid pUG23-Trs20) and BY4741 (isogenic cells containing the chromosomal copy of the Trs20 gene). (B) The temperature-sensitive phenotype of the Trs20 mutants is complemented by co-expression of a WT copy of Trs20 from the plasmid YCplac111. (C) The temperature-sensitive phenotype of the bet3-11 allele (plasmid YCplac111-bet3-11 in Δbet3 cells) is complemented by co-expressing a WT copy of Bet3 from the plasmid YCplac33. (D) Location of amino acid substitutions in pUG-trs20-1 and pUG-trs20-6. The secondary structure is of the mouse sedlin protein [19], but the amino acid positions refer to the yeast protein.
Figure 2.
GFP-Rer1p and GFP-Gos1p localization is unaffected in pUG-trs20-1 and pUG-trs20-6.
The indicated yeast strains transformed with (A) pSKY5 (that expresses GFP-tagged Rer1p) or (B) pUG34-Gos1 (that expresses GFP-tagged Gos1p) were observed by fluorescence microscopy at 23°C or after being shifted to 37°C for 30 minutes. In (A) white arrows indicate missorting of GFP-Rer1p to the vacuole in sec21-3 cells. In (B) the sec21-3 cells show both a punctate Golgi and a perinuclear ER fluorescence pattern (white arrows) at the restrictive temperature. (C) The indicated strains expressing GFP-Gos1p under control of the methionine-suppressible Met25 promoter were grown in medium containing 1 mM methionine (left panels), then washed and resuspended in methionine-free medium at 23°C (middle panels) or 37°C (right panels) for 30 minutes. (D) The indicated strains expressing GFP-Gos1p were grown in methionine-containing medium, washed and resuspended in methionine-free medium at 37°C for 30 minutes.
Figure 3.
Transport of CPY, ALP and Gap1p in the Trs20 mutants.
(A) CPY and ALP transport. Western blot analysis of CPY (top) and ALP (bottom) with total protein extracts from the indicated strains after 2 hours at 37°C. Mature, ER and Golgi forms of CPY are indicated by m, p1 and p2, respectively, and the ALP mature and precursor forms by mALP and proALP, respectively. (B) Fluorescence microscopy of the transmembrane protein GFP-Gap1(K9K16). Cells were grown in raffinose to log phase, galactose was added and the cells were then incubated at 27°C or at 37°C for 3 hours. (C) General secretion is not blocked in Trs20 mutants. Cells were grown to early log phase at 25°C, preincubated at 37°C for 30 minutes, radiolabeled for 15 minutes, and then chased for 30 min. The growth medium was separated from the cells by centrifugation, precipitated with TCA and separated by SDS-PAGE. Molecular weight markers (kDa) are indicated on the left.
Figure 4.
Trs20 mutants are hypersensitive to calcofluor white and mislocalize Snc1p.
(A) pUG-TRS20, pUG-trs20-1, pUG-trs20-6 and trs130-HAts cells were serially diluted and replica plated on YPD or YPD + 1 M sorbitol and incubated at 27°C or 37°C for 3 days or (B) on YPD or YPD containing 10 μg/ml of calcoflour white (CFW) and incubated at 27°C for 3 days. (C) The pUG-trs20 mutants mislocalize Snc1p. Early log phase cells expressing GFP-Snc1p were grown at 27°C or shifted to 37°C for 30 minutes. GFP-Snc1p is constitutively mislocalized in the pUG-trs20-6 mutant, similarly to Δtlg2 cells. The mislocalization can be rescued at 27°C and 37°C by co-expressing a WT copy of the Trs20 gene (YCplac111-TRS20). (D) A graph depicting the percentage of cells with mislocalized GFP-Snc1p, mean values ± SD, 3 replicates, n >100.
Figure 5.
Phenotypes of YCplac-trs20 mutants.
(A) Serial dilutions were spotted onto YPD plates or onto YPD containing 10 μg/ml of calcoflour white (CFW) and incubated for 3 days at the indicated temperatures. (B) Localization of GFP-Snc1p in the indicated strains. A graph depicting the percentage of cells with mislocalized GFP-Snc1p, mean values ± SD, 3 replicates, n >100. (C) YCplac-trs20 mutants do not undergo sporulation or meiosis. Light microscopy (left) and Hoescht staining (right) in each panel. In cells expressing a WT copy of Trs20, asci and the four products of meiosis are readily identified. No asci or meiosis were observed in the Trs20 mutants in sporulating cultures where the mother and daughter cell typically contain a single nucleus.
Figure 6.
Location of the studied mutations in the three-dimensional model of sedlin.
The model is based on the published structure of mouse sedlin [19], but amino acid numbers refer to the yeast Trs20 protein. (A) Location of mutated residues (yellow) in the hydrophobic core. (B) Location of mutated surface-exposed residues (green). The side chains of the glycine 108 residue (“G108”) are shown as the histidine residue in sedlin to mimic the tryptophan substitution in the G108W mutant (see text). (C) Detailed view showing the Asn147 residue of Trs20 (yellow) within the hydrophobic pocket of Bet3 [11]. (D) Surface rendition of the structure in (B). The residues contributing to the formation of the hydrophobic pocket [19] are shown in red. Dashed lines indicate the occupancy of the hydrophobic groove of Bet3 by Trs20 and of the groove A of Trs20 by Trs31. Visualization of the 3-D structure (accession number 1H3Q) was done using the MacPyMOL program.
Table 1.
Summary of phenotypes of Trs20 mutants.