THE CONSERVED G F MOTIF OF THE DNAJ CHAPERONE IS NECESSARY FOR THE ACTIVATION OF THE SUBSTRATE-BINDING PROPERTIES OF THE DNAK CHAPERONE/

The universally conserved DnaK and DnaJ molecular chaperone proteins b ind in a coordinate manner to protein substrates to prevent aggregatio n, to disaggregate proteins, or to regulate proper protein function. T o further examine their synergistic mechanism of action, we constructe d and characterized two DnaJ deletion proteins. One has an Il amino ac id internal deletion that spans amino acid residues 77-87 (DnaJ Delta 77-87) and the other amino acids 77-107 (DnaJ Delta 77-107). The DnaJ Delta 77-87 mutant protein, was normal in all respects analyzed. The D naJ Delta 77-107 mutant protein has its entire GIF (Gly/Phe) motif del eted. This motif is found in most, but not all DnaJ family members. In vivo DnaJ Delta 77-107 supported bacteriophage lambda growth, albeit at reduced levels, demonstrating that at least some protein function w as retained. However, DnaJP Delta 77-107 did not exhibit other wild ty pe properties, such as proper down-regulation of the heat-shock respon se, and had an overall poisoning effect of cell growth. The purified D naJ Delta 77-107 protein was shown to physically interact and stimulat e DnaK's ATPase activity at wild type levels, unlike the previously ch aracterized DnaJ259 point mutant (DnaJH33Q). Moreover, both DnaJ Delta 77-107 and DnaJ259 bound to substrate proteins, such as sigma(32), at similar affinities as DnaJ(+). However, DnaJ Delta 77-107 was found t o be largely defective in activating the ATP-dependent substrate bindi ng mode of DnaK, In vivo, the ability of the mutant DnaJ proteins to d own-regulate the heat-shock response was correlated only with their in vitro ability to activate DnaK to bind sigma(32), in an ATP-dependent manner, and not with their ability to bind sigma(32). We conclude, th at although the G/F motif of DnaJ does not directly participate in the stimulation of DnaK's ATPase activity, nevertheless, it is involved i n an important manner in modulating DnaK's substrate binding activity.