FIGURE 2. ApoB29 is membrane- and translocon-associated.

A, plasmid map of pSLW1-B29, a galactose-inducible, multicopy yeast apoB29 expression vector. B, both anti-HA and anti-apoB (1D1) antibodies detect apoB29, a ~160-kDa protein, expressed in yeast. Blots containing extracts from control (−) or apoB29-expressing (B29) cells were also probed for a ribosomal protein, L3, as a loading control. C, apoB29 fractionates with membranes after differential centrifugation of lysates from apoB29-expressing (B29) or control cells (−). Note the slower migrating band that nonspecifically cross-reacts with the HA antibody (marked with *). S1, 16,000 × g supernatant; P1, 16,000 × g pellet; S2, 150,000 × g supernatant; P2, 150,000 × g pellet. Protein levels were normalized by SDS-PAGE and Coomassie Brilliant Blue staining prior to Western blot analysis. D, apoB29 and the ER membrane-associated protein, Sec61p, migrate to a lower sucrose density when extracts from apoB-expressing cells are layered in a sucrose gradient. The soluble cytosolic proteins glucose-6-phosphate dehydrogenase (Met19p) and protein kinase C (Pkc1p) remain in the fractions where the extracts were loaded into the gradient. The lane marked E indicates 1% of the input (Load). A similar flotation pattern was observed for Sec61p, Met19p, and Pkc1p in cells transformed with the empty vector control (data not shown). E, apoB29 co-precipitates with Sec61p. Cell extracts were prepared from cells transformed with a vector control or pSLW1-B29 (B29) and were mock-treated or were incubated with anti-Sec61p or anti-Sec63p and protein A-Sepharose. The total proteins in the precipitates were resolved by SDS-PAGE and were immunoblotted with the indicated antisera. F, in the reciprocal experiment, Sec61p co-precipitates with apoB29. Cell extracts were prepared and incubated with anti-HA resin (+) or Sepharose 6B beads (−). The precipitates were immunoblotted with the indicated antisera.