Fus-Mid-GFP and

Abstract

<p><b>Pma1 localization is defective in </b><b><i>sec14^ts</i></b><b> cells and restored by expression of Sec14^G266D. </b><i>A, sec14^ts</i> cells expressing Fus-Mid-GFP and also containing empty vector, a vector expressing wild type Sec14 on a low copy plasmid, or Sec14^G266D 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. <i>B</i>, cells from <i>A</i> were quantified based on having only plasma membrane (PM) localization, only internal localization or both (vector n = 153, Sec14 n = 73, Sec14^G266D n = 107) <i>C</i>, the wild type <i>SEC14</i> gene was replaced with the <i>sec14^ts</i> 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 Sec14^G266D. 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.</p