Kar5p was contained within the ER/nuclear envelope  lumen. Protease protection was used to determined if Kar5p was  exposed to the cytoplasm or sequestered within the ER between  the inner and outer nuclear envelopes. Membrane fractions from  pheromone-induced cells (MS3987) were either treated with proteinase K or proteinase K with Triton X-100 for 0, 1, 2, 5, 10, and  20 min. After protease treatment, proteins were separated by  SDS gel electrophoresis, transferred to nitrocellulose, and probed  with anti-Kar5p, anti-Kar2p, or anti-Sec72p antibodies. Sec72p is  an ER protein exposed to the cytoplasm (Feldheim and Schekman, 1994), and Kar2p is an ER lumenal protein (Rose et al.,  1989). Sec72p was nearly entirely digested by protease after 20  min regardless of whether Triton X-100 was added. Kar2p and  Kar5p were protease protected in the absence of detergent for  >20 min. Kar2p and Kar5p were not inherently protease resistant  since neither was resistant to proteinase K when Triton X-100  was added; the solubilization of the nuclear membrane allowed  the protease access to lumen proteins. Thus, both Kar2p and  Kar5p are ER/nuclear envelope lumenal proteins.
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