Extension of Chronological Lifespan by Hexokinase Mutation in Kluyveromyces lactis Involves Increased Level of the Mitochondrial Chaperonin Hsp60
Figure 5
Analysis of involvement of Hsp60 in chronological aging and mitochondrial functionality. (a) Analysis by SDS-PAGE, western blotting of Hsp60 protein and northern analysis of HSP60 in the wt and in the isogenic rag5Δ strain. Determination of protein level by western analysis was performed as described in Section 2. Total mRNAs were prepared as described in Section 2 from cells grown on YPD medium for 24, 48 hr, 10 days. RNA was hybridized with labelled probes for HSP60 and 26S. Data are representative of three independent experiments, repeated with similar results. (b) Evaluation of gene dosage of HSP60 in determining the lifespan. Comparison of chronological survival in YPD medium of wt, rag5Δ strain, and the same strains overexpressing HSP60 gene. Survival was assessed in minimal medium for plasmid maintenance. Over time, aliquots of cultures were counted and serial dilutions were plated on solid medium. Colony forming units were counted after 3 days. Data are representative of three independent experiments, mean ± s.d. (c) DASPMI staining in wt cells overexpressing HSP60 gene after growth in YPD medium. Analysis was performed by taking aliquots at the indicated times and observing cells after DASPMI staining at the fluorescence microscopy. (d) Evaluation of mitochondrial nucleoids morphology along CLS in rag5Δ, wt and wt/HSP60 strains. Samples were collected at the indicated time-points and fluorescence was observed after DAPI in vivo staining.
