3 Results 52
3.2 Reconstitution of the Tim44:Ssc1 interaction cycle of the mitochondrial import
3.2.9 Ssc1 and Tim44 share complementary binding sites on matrix targeted pre-
targeted pre-proteins
Brownian ratchet mechanism posits that the vectorial translocation across TIM23 is driven by Ssc1 binding to substrates by preventing backsliding of pre-proteins to the cytosol (Ungermann et al., 1994). Earlier experiments have shown that a protein containing a large stretch of glutamates to which mtHsp70 cannot bind, can be imported efficiently (Okamoto et al., 2002). Stretches of negatively charged amino acids are known not to bind to Hsp70 group of chaperones and hence have a chance to slide back in the cytosol (Rudiger et al., 1997). This raises the question regarding the mechanism that prevents backsliding at non-Hsp70-interacting sequences. One possible solution might be the presence of other proteins at the TIM23 translocase that can bind transiently to these regions and prevent backsliding. Since Tim44 has been shown to be in the vicinity of preproteins in transit and interact with the unfolded polypeptides(Blom et al., 1993; Maarse et al., 1992; Rassow et al., 1994; Schneider et al., 1994), we sought to uncover if Tim44 binds to sequence specific regions within incoming preproteins. To
Figure 3.37: ATP or substrate induced dissociation of Tim44:Ssc1 is monophasic. Left and right panel: The dissociation of the Tim44:Ssc1 complex in presence of ATP (left panel) and P5 (right panel) was monitored by probing different distance vectors between Tim44 and Ssc1. The complexes were formed with donor labeled variants of Tim44 and acceptor labeled variants of Ssc1. The dissociation was initiated upon manually mixing either 2mM of ATP (left panel) or 6μM of P5 (right panel) and the recovery of donor fluorescence was obtained to follow the dissociation event. 2µM of Mge1 was present in buffer in order to facilitate ATP-charging of Ssc1 in subsequent step in case of ATP induced dissociation. The fluorescence recovery is normalized to compare the rate of increase in different distances.
83
understand the distribution of Ssc1-and Tim44-binding site on mitochondrial matrix targeted protein, scanning of peptide array of luciferase, a bona-fide substrate of Hsp70 group of chaperones was performed. The binding specificity observed for Ssc1 was mostly similar to the reported specificities of other Hsp70 chaperones like DnaK (Gragerov and Gottesman, 1994; Gragerov et al., 1994; Rudiger et al., 1997) (Figure 3.38 A, upper panel). Surprisingly, when peptide scanning of luciferase was performed to probe for Tim44 binding (Figure 3.38 B, upper panel), it was observed that Ssc1 and Tim44 share complementary binding sites on luciferase. To probe the generality of the complementary binding specificity of Tim44 and Ssc1 peptide array scanning of two authentic mitochondrial matrix proteins, mMDH and Hsp60 (Figure 3.38 A, middle and lower panel) was performed. Near exact complementary binding sites in all these proteins indicated that Ssc1 binds to hydrophobic amino acids surrounded by positively charged residues whereas Tim44 binds to peptide sequences rich in negatively charged amino acids which do not bind to Hsp70 group of chaperones (Figure 3.38 B, middle and lower panel).
This result hinted towards the possibility that Tim44 would bind to stretches of amino acids where Ssc1 is incapable of binding, further implying that Tim44 plays a role in preventing back sliding of proteins during translocation. Notably, the binding between Tim44 and the peptides could not be detected in solution phase (data not shown), indicating that the binding affinities between Tim44 and these peptides are extremely low further supporting the previous finding that Tim44 binds to unfolded polypeptides with low affinity (Schneider et al., 1994). Physiologically, the low affinity would be consistent with transient binding of substrates to Tim44 at the TIM23 translocase channel where a high affinity binding would abrogate translocation leading to a stalled translocase.
DnaK-like binding site specificity of Ssc1 also justified the use of DnaK binding site prediction algorithm to obtain Ssc1-binding sites, on mitochondrial-matrix-targeted pre- proteins, in order to calculate the physiological rate of Ssc1 cycling at TIM23 translocase.
84
In summary, the in vitro reconstitution of Tim44:Ssc1 interaction cycle and its kinetic analysis suggested that cycling of Ssc1 on Tim44 is driven by nucleotide bound to Ssc1, rather than the substrate. It was evident that the N-terminal domain of Tim44 and PBD of Ssc1 constitute the minimal binding domains of Tim44:Ssc1 complex, as probed by pull- down experiments and FRET based binding analyses. However, in the context of full length proteins both the domains of Tim44 were found to be spatially proximal to the PBD of Ssc1 and sub-domain Ia of NBD of Ssc1. It was also evident that the dissociation of Ssc1 from Tim44 is a single-step process ruling out the possibility of Tim44-anchored conformational change of Ssc1. Additionally, peptide-array scanning of authentic mitochondrial matrix targeted proteins revealed the near exact complimentarity of binding sites of Ssc1 and Tim44 on the precursors.
Figure 3.38:Peptide scans with purified Ssc1 and Tim44. Cellulose bound peptide scans derived from the sequences of Firefly-luciferase(top), Sc mitochondrial MDH(middle), Sc Hsp60 (bottom) were screened for binding with Ssc1(A) and Tim44(B). Last spot of each row is indicated with the number.
85
This result together with weak binding affinity of Tim44 to non-Hsp70 binding sites might explain how the backsliding of preproteins is prevented at the translocase channel. All the data can only be reconciled in the context of Brownian ratchet model of protein import motor.