Figure 4
Gykl1 and Gk2 target to the outer membranes of the mitochondria through their unique C-terminal domains. (a) HEK293T cells transiently expressing various GFP-tagged proteins were stained with the MitoTracker dye to visualize the mitochondria. Arrows indicate areas of mitochondrial clustering. Hoechst 33342 was used to visualize the nuclei. Scale bar: 10 μm. (b) HEK293T cells transiently expressing GFP-tagged proteins were fractionated and blotted with antibodies against GFP. WCE, whole-cell extract. Cyto, cytoplasmic fraction, Mito, mitochondrial fraction. Antibodies against GAPDH and TOM20 served as loading control for the cytosolic and mitochondrial fractions respectively. (c) HEK293T cells transiently expressing HA-tagged Gykl1 and Gk2 were used to isolate mitochondrial fractions. The fractions were treated with proteinase K in the presence or absence of Triton X-100 before being immunoblotted with anti-HA antibodies. The antibody against TOM20, a mitochondria outer membrane protein, served as a positive control. (d) The C-terminal regions of Gyk-like genes from different species are aligned. Testis-specific genes appear to contain additional sequences in their C-termini compared with Gyk orthologues that are not restricted to testis. (e) HEK293T cells transiently expressing GFP-tagged Gyk as well as full-length and truncation mutants of Gykl1 were stained with the MitoTracker dye to visualize the mitochondria, and examined under a fluorescent microscope. Hoechst 33342 was used to visualize the nuclei. Arrows indicate areas of mitochondrial clustering. Scale bar, 10 μm.
