Bag-1M Accelerates Nucleotide Release for Human Hsc70 and Hsp70 and Can Act Concentration-dependent as Positive and Negative Cofactor

Abstract

The cytosol of mammalian cells contains several Hsp70 chaperones and an arsenal of cochaperones, including the anti-apoptotic Bag-1M protein, which regulate the activities of Hsp70s by controlling their ATPase cycles. To elucidate the regulatory function of Bag-1M, we determined its influence on nucleotide exchange, substrate release, ATPase rate, and chaperone activity of the housekeeping Hsc70 and stress-inducible Hsp70 homologs of humans. Bag-1M and a C-terminal fragment of it are potent nucleotide exchange factors as they stimulated the ADP dissociation rate of Hsc70 and Hsp70 up to 900-fold. The N-terminal domain of Bag-1M decreased the affinity of Bag-1M for Hsc70/Hsp70 by 4-fold, indicating a modulating role of the N terminus in Bag-1M action as nucleotideexchangefactor.Bag-1MinhibitedHsc70/Hsp70-dependent refolding of luciferase in the absence of P i . Surprisingly, under physiological conditions, i.e. low Bag-1M concentrations and presence of P i , Bag-1M activates the chaperone action of Hsc70/Hsp70 in luciferase refolding. Bag-1M accelerated ATP-triggered substrate release by Hsc70/Hsp70. We propose that Bag-1M acts as substrate discharging factor for Hsc70 and Hsp70.The Hsp70 proteins are involved in folding processes throughout the entire life span of proteins. The molecular basis of their functions is the transient interaction with substrates through ATP-controlled cycles (1, 2). In the ATP state Hsp70s have a low affinity for substrates, and in the ADP state the affinity for substrates is high (3, 4). ATP hydrolysis was shown to be stimulated synergistically by the simultaneous interaction with a substrate and a cochaperone of the DnaJ family (5-8). Under physiological conditions nucleotide exchange is rate-limiting for substrate release (3, 9, 10). For this functional cycle the regulation by cochaperones is an important issue. How do they act in the ATPase cycle and do they exhibit specificity for a given Hsp70 partner? Significant influence on the ATPase cycle of Hsp70s was shown for members of the families of DnaJ and Bag proteins.The Bag family of proteins consists of six members that share a conserved sequence stretch of 40 -50 residues at the C terminus (11,12). The C terminus of Bag-1 was also shown to be responsible for the interaction with the ATPase domain of Hsp70 proteins (11, 13-16). The anti-apoptotic protein Bag-1 that exists in at least three translational initiation variants, Bag-1L, Bag-1M, and Bag-1S, received the most attention. Bag-1M was reported to induce ADP release by Hsc70 (13) similar to GrpE, the nucleotide exchange factor for the DnaK-type Hsp70 homologs of bacteria. However, such an analogous function for GrpE was discussed controversially as Bag-1M did not accelerate the dissociation of Hsp70⅐ADP or Hsp70⅐ATP complexes (17). Whether Bag-1M inhibited or stimulated the refolding of denatured substrates by Hsp70s in vitro and in vivo was also controversial (15,(17)(18)(19). All studies agree that Bag-1M stimulates the steady-state ATPase activity of Hsp70 and Hsc70. How...

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