SUBCELLULAR-LOCALIZATION AND CHAPERONE ACTIVITIES OF BORRELIA-BURGDORFERI HSP60 AND HSP70

Subcellular locations and chaperone functions of Hsp60 and Hsp70 with flagellin were investigated in Borrelia burgdorferi. Sodium dodecyl su lfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis of fractionated cells showed Hsp60 to be present in the solu ble fractions and the Triton X-100 detergent-soluble membrane fraction at growth temperatures ranging from 20 to 37 degrees C. The relative amount of Hsp60 associated with the membrane increased with growth tem perature. Hsp70 was found in soluble fractions at growth temperatures between 28 and 37 degrees C, but at 20 degrees C it was also present i n the Triton X-100-insoluble membrane fraction. Immunoelectron microsc opy revealed that the majority of Hsp60 was localized in the cytoplasm but a detectable fraction (similar to 30%) was associated with the ce ll envelope. The chaperone functions of Hsp60 and Hsp70 were analyzed by immunoprecipitation of [S-35]methionine-labeled cell lysates under nondenaturing conditions in the presence or absence of ATP. Hsp70 was found to bind flagellin at all temperatures tested between 33 and 41 d egrees C. This association could be decreased with ATP when cells had been incubated at 41 degrees C during radioactive labeling but not at lower temperatures. Both flagellin and Hsp70 were found to associate w ith Hsp60, forming a complex of the three proteins. Hsp70 association with this complex could be decreased with ATP, but flagellin binding t o Hsp60 was ATP independent at all temperatures studied. Both Hsp70 an d flagellin were inaccessible to monoclonal antibodies against them wh en bound to Hsp60. These studies suggest that in B. burgdorferi, a maj or function of Hsp60 and Hsp70 is in the molecular processing of flage llin.