The Evolution and Function of Co-Chaperones in Mitochondria - The Networking of Chaperones by Co-chaperones - page 204

Disruption of heterodimer formation was observed when residues in the con-
served “arm” region, and the J-domain of Pam18, and C-terminal region containing
the J-like domain of Pam16 were mutated or deleted. In all cases, these disruptions
were either lethal or severely affected the rates of protein import and cell growth
(D'Silva et al. 2005 , 2008 ; Hayashi et al. 2011 ; Pais et al. 2011 ). While expression
of the C-terminal domain of Pam16 alone can complement the depletion of the
wild-type protein, this region can be substituted by the J domain of Pam18, but
under those circumstances the N-terminal domain of Pam16 becomes indispens-
able (D'Silva et al. 2008 ; Mokranjac et al. 2006 , 2007 ). These findings demonstrate
that the interaction of the J and J-like domains of Pam18 and Pam16 is functionally
indispensable for the protein import process. Interestingly, this type of interaction
between co-chaperones has not been observed in any other Hsp70 system.
In in vitro assays, the Pam18/Pam16 heterodimer stimulates the ATPase activity
of Ssc1 to a significantly lesser degree than Pam18 alone (Li et al. 2004 ; Mokranjac
et al. 2006 ; Chacinska et al. 2005 ). This finding led to the proposal that Pam16 acts
as regulator of Pam18, preventing it from interacting with Ssc1 in the absence of the
substrate, i.e. preventing the unnecessary activity of the protein import motor. Reg-
ulation of the Pam18 activity, under this model, is mediated by the changes in the
conformation of the heterodimer, specifically the J/J-like domain interface, which
alternates between the Ssc1 stimulating form and the inactive form (Endo et al.
2011 ; Mokranjac and Neupert 2010 ; van der Laan et al. 2010 ). However, it remains
unclear whether the reduction in the ATPase stimulating activity of the heterodimer
plays an important role in vivo . Mutations in the Pam18 J domain that reduce its
ATPase stimulating activity were deleterious when the “arm” region (critical for
binding to Pam16) was also disrupted. ATPase activity enhancing mutations in the
J domain alleviated the effects of mutations that destabilize the heterodimer forma-
tion (Pais et al. 2011 ). It is possible that in vivo , the heterodimer can stimulate Ssc1
sufficiently for the ATP hydrolysis to occur, especially considering the relatively
high effective concentration of these proteins at the import site.
In yeast, a duplicate gene ( MDJ2 ) encodes a paralogue of Pam18 (Westermann
and Neupert 1997 ; Mokranjac et al. 2005 ). It is not clear if there is any functional
advantage gained from this isoform of Pam18, arisen in the common ancestor of
Saccharomycotina (Hayashi et al. 2011 ). There are informative differences in the
way the two isoforms of Pam18 perform act. Although the Mdj2 isoform interacts
with Pam16, the latter does not antagonize, but rather enhances the Mdj2 mediated
stimulation of ATP hydrolysis by Ssc1 (Mokranjac et al. 2005 ). Furthermore, while
Mdj2 can act in the protein import motor in place of Pam18, it cannot fully substi-
tute its function under anaerobic conditions in vivo (Hayashi et al. 2011 ).
Pam18 is associated with the TIM23 complex during the protein import reac-
tion. The key role of Pam16 is to mediate this interaction (Kozany et al. 2004 ;
Li et al. 2004 ). When the Pam18/Pam16 interaction is disrupted by site directed
mutagenesis targeting either protein, Pam18's binding to the TIM23 complex is re-
duced (D'Silva et al. 2008 ). The same effect is observed when Pam16's N-terminal
domain was deleted, suggesting that Pam16 binds to the TIM23 complex via this
domain (Mokranjac et al. 2007 ). The two N-terminal domains of Pam18 are also