Figure 5.

Yeast Taz1p harboring authentic BTHS mutations that occur in the putative interfacial membrane anchor of Taz1p localize to mitochondria, but are nonfunctional. (A) ClustalW alignment of the putative interfacial membrane anchor (boxed) of Taz1p from human, mouse, and S. cerevisiae. The BTHS mutations are indicated at the top, with mutations occurring at conserved and identical residues provided in green and red, respectively. (B) The relative expression of the four different BTHS mutants (three clones/mutant) was determined from whole-cell extracts by immunoblotting for Taz1p (bottom) with KDH serving as a loading control (top). (C) The same as B, except that three clones derived from a humanized yeast Taz1p were analyzed next to the three Taz1p mutants harboring single BTHS mutations occurring at conserved residues. (D and E) Steady-state ³²P labeling and analyses were performed as described in Fig. 3 (B and C). Δtaz1 yeast accumulate significant amounts of MLCL relative to wt yeast (P ≤ 0.001), as determined by t-test. All of the BTHS mutants, with the notable exception of the humanized Taz1p, demonstrate a statistically significant accumulation of MLCL relative to Δtaz1 transformed with wt Taz1p ([WT Taz1p]; P < 0.001) as determined by one-way ANOVA, with Holm–Sidak pairwise comparisons. Mean ± SEM. n = 4, except for humanized Taz1p, where n = 3. (F) Fractions were prepared from the indicated yeast strains through a series of differential centrifugations. 50 μg of each fraction was separated by SDS-PAGE and analyzed by immunoblot using antisera specific for the indicated subcellular organelle. n = 2.