Figure 7.

Sec35p is a peripheral membrane protein. (a) Sec35p partitions into soluble and membrane-associated pools. A wild-type strain was grown to logarithmic phase, lysed with glass beads, and then centrifuged at 1,000 g for 3 min to generate the S1 supernatant fraction. The S1 supernatant was centrifuged at 175,000 g to obtain the S175 supernatant and P175 pellet fractions. (b) The membrane-associated portion of Sec35p fractionates similarly to the Golgi protein, Sed5p. The S1 supernatant was centrifuged at 10,000 g to generate the S10 supernatant and P10 pellet fractions, and the S10 was subsequently centrifuged at 175,000 g to obtain the S175 supernatant and P175 pellet fractions. (c) Sec35p behaves as a peripheral membrane protein. The S1 supernatant was incubated with buffer 88, 1% Triton X-100, 1 M NaCl, or 100 mM Na₂CO₃, pH 11, centrifuged at 175,000 g, and separated into supernatant and pellet fractions. (a–c) Aliquots of each fraction were separated by SDS-12%PAGE and immunoblotted with affinity-purified anti-Sec35p (top panel), anti-Sed5p (center panel), or anti-PGK (bottom panel) antibodies. The samples loaded in each lane were derived from equivalent amounts of starting material.