FIGURE 4.
RTA is retained in the membrane fraction when CDC48-dependent retro-translocation is impaired. A, protoplasts were transfected with pDHA vector alone (vector) or the RTAE177D-encoding plasmid plus wtCDC48 or CDC48QQ plasmids. Protoplasts were radiolabeled with [35S]cysteine and [35S]methionine for 6 h, before being homogenized in the absence of detergent and fractionated to yield microsomes (M) and cytosol (C). Proteins were immunoselected sequentially using anti-RTA and anti-BiP antisera, and analyzed by reducing SDS-PAGE and fluorography. Numbers on the left indicate molecular mass markers in kilodaltons. B, protoplasts were transfected with the plasmids shown, before being radiolabeled for 1 h and chased as indicated. The cells were then homogenized as in A, before dividing three ways and incubating in the absence or presence of proteinase K (PK) and detergent (Triton X-100). RTA was immunoselected and resolved as in A. The position of the 30-kDa molecular mass marker is indicated on the left. C, quantitation, from three separate pulse-chase experiments, of the amount of RTA made in the absence or presence of CDC48QQ that was subsequently protected from proteinase K after 3 h. Bars indicate standard deviation and different letters above the bars indicate a significant difference (p < 0.05).