Figure 8.

Complementation of Yeast Mutant K616 through Expression of SCA1p nt85.

(A) Complementation of yeast Ca²⁺-ATPases mutant by an N-terminal–truncated mutant pump (SCA1p nt85). Wild-type (W303-1A) and triple mutant (K616) cells were transformed with a vector (pYES2) alone, or with pYES2-SCA1 and pYES2-SCA1 nt85. The cells were streaked onto SC-URA plates containing 10 mM EGTA, pH 6.0, and either glucose (Glc) or galactose (Gal); the cells were incubated for 4 days at 30°C. A diagram indicates yeast strains and transformed vectors. Full-length and ΔN indicate SCA1p and SCA1p nt85, respectively.

(B) Sucrose gradient fractionation of SCA1p and SCA1p nt85. Microsomal membranes isolated from yeasts transformed with pYES2-SCA1 and pYES2-SCA1 nt85 were fractionated over a continuous sucrose gradient of 20 to 60% (w/w), and 1-mL fractions were collected from the top of each gradient. Samples (10 μL) from each gradient fraction were subjected to 8% SDS-PAGE and transferred to Immobilon-P membranes. The gel blots were probed with purified anti-SCA1 antibody. Immunoreactive bands were detected by electrochemiluminescence and exposure to x-ray film. Asterisks indicate the peak fractions for plasma membrane fractions, as determined by assays for vanadate-sensitive (calcium-independent) H⁺-ATPase activity.