Localization of TEF30 in thylakoids of C. reinhardtii wild-type and mutant cells. A, Salt treatments during cell fractionation. Whole cells (WC) of strain cw15-325 were resuspended in lysis buffer (LB) containing the indicated salts, subjected to freezing/thawing, and separated into soluble (S) and pellet (P) fractions. One sample resuspended in lysis buffer only was subjected to sonication instead of freezing/thawing. Proteins were separated by SDS-PAGE and immunodetected with antibodies against TEF30 and against integral membrane protein cytochrome f (Cytf) and stromal CGE1 as controls. B, Trypsin treatment of thylakoid membranes. Isolated thylakoids from strain cw15-325 were incubated on ice with trypsin, separated by SDS-PAGE, and immunodetected with antibodies against TEF30 and the peripheral CF1β subunit of the ATPase complex, lumenal plastocyanin (PCY1), and integral membrane protein cytochrome f as controls. C, Cell fractionation of various photosynthesis mutants. Whole cells of the wild type (WT) and thylakoid membrane protein mutants were separated into soluble and pellet fractions via freezing/thawing. Proteins were separated by SDS-PAGE and immunodetected with antibodies against TEF30 and against integral membrane protein cytochrome f or D1 and stromal CGE1 as controls.