FIGURE 4.

hssSPTs lacking the second predicted TMD retain their native membrane topology. A, sealed microsomal vesicles were prepared from yeast expressing hLCB1, hLCB2a, and N-terminally HA-tagged ssSPTa with either the C-terminal 21 or 25 amino acids replaced by a Myc tag. The microsomes were mock digested or digested with proteinase K (PK) in the absence or presence of Triton X-100. The luminal ER protein, Kar2p, served as a control for vesicle integrity. B, the C-terminal 21 or 25 amino acids of ssSPTa were replaced with a GC cassette. After expression in yeast, glycosylation was assessed as described in Fig. 2.