Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60

Abstract

Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.

Figure 1. Mortalin and HSP60 binding in vivo

(A) Mortalin and HSP60 were repeatedly precipitated with respective specific polyclonal antibodies, as indicated. Co-precipitated mortalin protein in HSP60 immunocomplexes and HSP60 in mortalin immunocomplexes were detected by Western blotting with specific monoclonal antibodies. Note that some mortalin co-precipitated with HSP60 (lanes 1–4), and similarly some HSP60 co-precipitated with mortalin (lanes 5–8). Rabbit IgG was used as a negative control (lanes 9–11). Detection of mortalin and HSP60 in immunodepleted lysates showed some decrease of mortalin in HSP60-immunodepleted lysates, and vice versa (lanes 12–15). (B) GST-tagged mortalin was mixed with cell lysates from which HSP60 was immunoprecipitated. Co-precipitated GST-tagged mortalin was detected by Western blotting with anti-GST antibody. Note that in lane 1, no GST-mortalin was added. Thus the control antibody pulled-down unspecific proteins (molecular masses close to GST–mortalin; lanes 1, 2, 5 and 6) that cross-reacted with anti-GST antibody. GST-mortalin (mot-2) co-precipitated with HSP60, but not with isotype matched control antibody. Supernatant after immunoprecipitation of HSP60 was also run on a gel to confirm the presence of GST–mortalin in control precipitation. (C and D) GST-tagged deletion mutants of mortalin were added to the cell lysates, following which HSP60 was immunoprecipitated. Whereas full-length mortalin (C) and its mutants containing amino acid residues 105–435 (C) or 105–252 (D) immunoprecipitated with HSP60, neither GST alone nor residues 250–435 (D) or 403–679 (C) showed binding.

Figure 2. Double immunostaining of mortalin and HSP60

Co-localization of mortalin and HSP60 in human normal embryonic-lung fibroblasts (MRC-5) and osteosarcoma (U2OS) cells with Qdot bioconjugates. Note that the two proteins overlap by approx. 70% (shown as the yellow colour). Distinct red and green staining was visible both in normal and transformed cells.

Figure 3. HSP60 knock-down causes growth arrest of osteocarcinoma cells

(A) Expression levels of HSP60 subsequent to the stable transfections of indicated shRNA expression plasmid in U2OS cells as examined by Western blotting are shown. Target sites 102 and 160 were better than 335 and 712. The morphology (B) and colony-forming efficiency (C) of vector and HSP60 shRNA expression plasmid-transfected U2OS cells are shown.

Figure 4. Lifespan extension of human fibroblasts

Overexpression of mortalin, but not HSP60, causes lifespan extension of human fibroblasts (A). Vector and HSP60-transfected cells phased out at 59 PDs, and did not show any increase in cell number over the next 4 weeks. Mortalin-transfected cells underwent 71 PDs in two independent experiments. (B) Induction of senescence in U2OS cells by exogenous expression of p14^ARF causes a decrease in mortalin, but not in HSP60. Actin was used as an internal loading control, and p53 was used as a downstream marker for p14^ARF activity.