Figure 8.

MA3* increases MA-GFP-PM binding, whereas ESCRT deletion does not.A and B, WT yeast cells or the indicated ESCRT mutants expressed MA-GFP or MA3*-GFP from a 2µ vector with induced MET3 promoter. A, MA-GFP binding to the PM was analyzed by fluorescence microscopy, showing PM rim staining for MA3*. DIC, differential interference contrast. B, membrane-containing 232,000 × g pellets (P) and cytosol-containing supernatants (S) derived from cell extracts (T) were analyzed by immunoblotting with anti-GFP antibodies, showing that increased MA3* amounts sediment compared with MA, whereas similar MA amounts sediment from WT and ESCRT mutant cell extracts. The pellet samples were 5× concentrated compared with the supernatant and extract samples. The cytosolic protein PGK and the integral ER membrane protein Sec61 served as references.