ENHANCEMENT IN AMOUNT OF P1 (HSP60) IN MUTANTS OF CHINESE-HAMSTER OVARY (CHO-K1) CELLS EXHIBITING INCREASES IN THE A-SYSTEM OF AMINO-ACID-TRANSPORT

Mutants of CHO-K1 cells with varied levels of A system activity, proba bly the result of increases in absolute amount of the A system transpo rter, have corresponding increases in levels of peptides banding at 62 -66 and 29 kDa. Mutant ala(r)4-H3.9, showing the highest increase of A system activity and of 62- to 66- and 29-kDa peptides, was selected f or this study. The N terminus 16-amino acid sequence of the 62- to 66- kDa peptide(s) of this mutant showed between 80% and 100% identity wit h the mammalian mitochondrial 60-kDa heat shock protein P1 (hsp60). Tw o-dimensional gel electrophoresis of the 62- to 66-kDa band showed two major, a minor, and several smaller spots (of same mass but different pI values) for both wild type (WT) and mutant, with the two major spo ts being of greater density in the mutant. Immunoblots with antibody t o P1 identified the two major and minor peptides as P1 related. Two-di mensional gels of whole cell extracts of the WT and ala(r)4-H3.9 confi rmed these findings and indicated that the two major bands of the muta nt were 2.4 times as abundant as that found for the WT. A plasma membr ane fraction of the mutant, exhibiting 4.8 times more A system activit y than the WT, contained 3.6 times as much P1 as the WT. Immunoblots w ith antibodies to P1, mitochondrial malate dehydrogenase, and to the m itochondrial F1/F0-ATPase demonstrated that the increased amount of P1 observed in the mutant was not the result of increases in amount of m itochondrial protein. Northern blot analysis demonstrated that the mut ant had 2.5 times as much mRNA for P1 as the WT. The close analogy wit h the relationship between A system and Na+,K+-ATPase suggests that th ere is a coordinate regulation of the A system of amino acid transport , Na+,K+-ATPase, and P1 protein, probably as a result of mutation in a shared regulatory element. The possible role of P1 in A system functi on is discussed.