Table 1.
sec14ts suppressing gene deletions.
Figure 1.
The Sec14 G266D protein can provide the essential function of Sec14.
Serial dilutions of cells cultured at 25°C and then plated and grown at 25°C and 37°C for 3 days. A, wild type, sec14ts and sec14ts rpn4Δ. B, The sec14ts strain transformed with either empty vector, a plasmid carried at low copy (ARS/CEN) containing wild type Sec14, or a high copy (2 μ) plasmid containing Sec14G266D.
Figure 2.
Effect of temperature on Sec14 and Sec14 G266D protein levels.
A, the sec14ts cells were transformed with a plasmid expressing Sec14 containing an N-terminal T7 epitope, untagged Sec14, or empty vector. SEC14 expression was driven by the constitutive GPD1 promoter. Cells were grown in solution at 25°C to mid-logarithmic phase, and serial dilutions of identical numbers of cells were spotted onto plates and incubated at 37°C for two days. B, cells expressing T7-Sec14 or Sec14G266D were grown to mid-logarithmic phase at 25°C, with a subset shifted to 37°C for 2 hrs. Cells were disrupted by three passes through a French press and membranes were separated from soluble proteins by differential centrifugation. Proteins in each fraction were separated by SDS-PAGE, transferred to PVDF membrane, and western blots were performed. In the blots shown 10 fold more protein extract was loaded in each Sec14G266D lane compared to extracts containing wild type Sec14 for blots versus the T7 epitope due to protein expression level differences.
Figure 3.
The levels of Sec14G266D is regulated by the proteosome.
A, the level of Sec14 and Sec14G266D in sec14ts rpn4Δ cells. B, the level of Sec14 and Sec14G266D in sec14ts cells treated with MG132. Strains were transformed with plasmids expressing Sec14 or Sec14G266D containing an N-terminal T7 epitope tag and were grown to mid-logarithmic phase at 25°C, with a subset shifted to 37°C for 2 hours (A). For MG132 treatment cells were grown as before and shifted to 37°C in the presence of 100 μM MG132 for 2 hours. Cells were disrupted by three passes through a French press and unbroken cells removed by centrifugation. Protein extract was separated by SDS-PAGE, transferred to PVDF membrane, and western blots versus the T7 epitope were performed. Pgk1 was used as load control.
Figure 4.
Known vesicular trafficking pathways are still aberrant in growing cells expressing Sec14G266D.
The sec14ts strain transformed with either empty vector, a plasmid carried at low copy (ARS/CEN) containing wild type Sec14, or a high copy (2 μ) plasmid containing Sec14G266D were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 1 hr subsequent to determination of: A, invertase secretion (mean ± SE of three separate experiments performed in duplicate), B, or internal retention of Bgl2 at 2 and 16 hrs, similar results were seen at both time point with the 2 hr time point shown. C, the sec14ts strain containing plasmid borne GFP-Snc1 was transformed with either empty vector, a plasmid carried at low copy (ARS/CEN) containing wild type Sec14, or a high copy (2 μ) plasmid containing Sec14G266D. Cells were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 2 hrs. The localization of GFP-Snc1 was determined by fluorescence microscopy in live cells. D, The strains were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 15 min prior to the addition of FM4-64. The trafficking of FM4-64 in live cells was visualized by fluorescence microscopy.
Figure 5.
Membranes accumulate in growing cells expressing Sec14G266D.
Wild type cells and sec14ts cells containing empty vector, a vector expressing wild type Sec14 on a low copy plasmid, or Sec14G266D on a high copy (2 μ) plasmid, were grown at 25°C to mid-logarithmic phase and an aliquot transferred to 37°C for 1 hr followed by incubation in 1.5% KMnO4, 1% sodium periodate, and then 1% NH4Cl subsequent to embedding and viewing by transmission electron microscopy.
Figure 6.
Pma1 localization is defective in sec14ts cells and restored by expression of Sec14G266D. A, sec14ts cells expressing Fus-Mid-GFP and also containing empty vector, a vector expressing wild type Sec14 on a low copy plasmid, or Sec14G266D on a high copy (2 μ) plasmid, were grown at 25°C in 1% raffinose containing medium to mid-logarithmic phase. Cells were shifted to 37°C in pre-warmed 2% galactose containing medium for 3 hours. B, cells from A were quantified based on having only plasma membrane (PM) localization, only internal localization or both (vector n = 153, Sec14 n = 73, Sec14G266D n = 107) C, the wild type SEC14 gene was replaced with the sec14ts allele in a yeast strain expressing chimeric Pma1-RFP. The strain was transformed with either empty vector, a plasmid carried at low copy (ARS/CEN) containing wild type Sec14, and low and high copy (2 μ) plasmids containing Sec14G266D. Cells were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 16 hrs subsequent to determination Pma1-RFP localization by fluorescence microscopy.
Table 2.
Yeast strains used in this study.