FIGURE 2.

SenP5 translocates to the mitochondrial membrane during the G₂/M phase. A, COS-7 cells were transfected with OCT-CFP, SenP5-YFP, fixed, and stained with Hoechst, prior to analysis by fluorescence microscopy. B, mitotic cells at prophase, anaphase, and telophase, showing SenP5-YFP on the mitochondria (red). Chromatin distribution was revealed by Hoechst (green). Scale is 5 μm. C, COS-7 cells were synchronized with either a double thymidine block alone (G₁/S), or the thymidine block followed by a 12-h release in 100 ng/ml nocodazole (G₂/M). These cells were harvested, and total extracts, cytosolic and mitochondrial fractions were obtained. Then, 25 μg of protein from each fraction was loaded on the gel and processed for electrophoresis and Western analysis with antibodies against endogenous SenP5, DRP1, HSP60, TOM20, and HSP70, as shown. Cyclin A (G₁/S) and cyclin B1 (G₂/M) were probed as synchronization controls. Asterisks indicate the higher migrating form of DRP1 that is lost upon the delivery of SenP5 to the mitochondria in nocodazole-treated cells. D, COS-7 cells were synchronized with double thymidine block, followed by a 6-h release to enrich endogenous SenP5 on the mitochondria. Cells were harvested, mitochondria were isolated, and 20-μg aliquots were either left untreated (lane 1), or treated with 0.1 mg/ml trypsin in the presence or absence of 1% Triton X-100 (second and third lanes 2). Western blots were probed for endogenous SenP5, and cytochrome c as an internal control.