J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix

Abstract

The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel. Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate. Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function. However, a previously unstudied essential gene, PAM18, encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane. Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix. We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1's function in protein import. M ost proteins of the mitochondrial matrix are synthesized on cytosolic ribosomes and must therefore be imported across the outer and inner mitochondrial membranes. Translocation across the inner membrane occurs through the inner membrane channel and is driven by the membrane potential and an import motor (1-3). Three critical components of this motor are the major Hsp70 molecular chaperone of mitochondria (mtHsp70; Ssc1 in yeast); the peripheral inner membrane protein Tim44, which tethers Ssc1 to the import channel; and the nucleotide release factor for Ssc1, Mge1. Multiple cycles of Ssc1 binding to and release from translocating polypeptide, driving the import process, are required for import of proteins into the mitochondrial matrix (1-3).Ssc1, like other Hsp70s, contains a C-terminal domain that binds short segments of unfolded polypeptides rich in hydrophobic amino acids (4, 5). The N-terminal ATPase domain regulates this binding through interaction with adenine nucleotides. In turn, binding of the peptide segment to the C-terminal domain stimulates the ATPase activity of the N terminus. In the ATP-bound state, interaction with peptide substrate is unstable, with very fast on and off rates. In the ADP-bound state, the interaction is relatively stable, with slow on and off rates. Therefore, it is thought that the ATP-bound form of Hsp70 initiates interaction with polypeptide substrate, which is then stabilized by the hydrolysis of ATP. Exchange of ATP for bound ADP results in the release of peptide, thus completing the cycle.Hsp70s rarely, if ever, function independently. Rather, they function with cochaperones. J proteins, named because of the presence of the signature J domain that interacts with the ATPase domain, stimulate Hsp70's ATPase activity, thus facilitating interaction with substrate polypeptides (6, 7). This activity is a critical feature of J...

The Presequence Translocase-associated Protein Import Motor of Mitochondria

“…The Pam16 domain possesses a similar length and predicted secondary structure as the Pam18 domain, but the characteristic sequence motif HPD (His-Pro-Asp) that is strictly conserved in all known Jproteins is missing in Pam16. As expected for a protein of the DnaJ family, the purified J-domain of Pam18 stimulated the ATPase activity of mtHsp70 (28,29). Initial studies with purified full-length Pam16 did not show a stimulatory effect on the ATPase activity of mtHsp70 (31,32).…”

“…This was in contrast to the strong stimulation of the ATPase activity of mtHsp70 by the purified Jdomain of Pam18, termed Pam18 J (Fig. 1B) (28,29). We then asked if Pam16 S enhanced the stimulating activity of Pam18 J by adding both proteins together.…”

Role of Pam16's degenerate J domain in protein import across the mitochondrial inner membrane

“…Like any typical Hsp70, Ssc1 functions in the import process with two cochaperones that facilitate its reaction cycle. Pam18 (Tim14) serves as its J protein partner, stimulating ATP hydrolysis and thus, stabilizing the interaction with translocating polypeptide (10)(11)(12). Pam18, like all J proteins, contains a J domain with a conserved histidine, proline, aspartic acid (HPD) tripeptide that is essential for function (10)(11)(12).…”

“…Pam18 (Tim14) serves as its J protein partner, stimulating ATP hydrolysis and thus, stabilizing the interaction with translocating polypeptide (10)(11)(12). Pam18, like all J proteins, contains a J domain with a conserved histidine, proline, aspartic acid (HPD) tripeptide that is essential for function (10)(11)(12). Mge1 is a nucleotide release factor that facilitates release of ADP and rebinding of ATP, thus destabilizing the polypeptide interaction (8,13,14).…”

Remodelling of the active presequence translocase drives motor-dependent mitochondrial protein translocation

“…For both activities, the soluble co-chaperone Mge1 stimulates ADP/ATP exchange 9,10 . Although matrix co-chaperones act as J-proteins stimulating the ATPase activity of mtHsp70 during protein folding, a TIM23 complex-associated membrane integral J-protein (Pam18) is import specific [11][12][13][14][15] . At the resting translocase, Pam18 forms a complex with the J-like Pam16.…”