The Evolution and Function of Co-Chaperones in Mitochondria - The Networking of Chaperones by Co-chaperones - page 205

involved in interaction of the heterodimer with this complex. Disruption of either
the intermembrane space or the transmembrane domains of Pam18 weaken this
association (Hayashi et al. 2011 ; Mokranjac et al. 2007 ; Chacinska et al. 2005 ;
D'Silva et al. 2008 ). The intermembrane space domain likely binds directly to the
C-terminal region of Tim17, a component of the TIM23 complex (Chacinska et al.
2005 ; Schilke et al. 2012 ). Simultaneous disruption of the Pam16 and Pam18 medi-
ated interactions of the heterodimer with the TIM23 complex is lethal to yeast cells
(Schilke et al. 2012 ).
Two further mitochondrial matrix proteins regulate the interaction of the Pam18/
Pam16 heterodimer with the TIM23 complex, Tim44 and Pam17. The peripheral
membrane located protein Tim44 was the first co-chaperone component of the pro-
tein import machinery to be identified (Maarse et al. 1992 ; Scherer et al. 1992 ;
Weiss et al. 1999 ). It has a matrix-exposed segment with limited similarity to a J
domain, mostly in the second helix of the canonical fold. This segment of the pro-
tein does not contain the catalytic HPD motif, but is essential for Tim44 function
(Merlin et al. 1999 ; Rassow et al. 1999 , 1994 ). The three-dimensional structure of
the C-terminal domain of Tim44 revealed a large hydrophobic pocket that was pro-
posed to mediate binding to the mitochondrial inner membrane. Subsequent in vitro
studies have demonstrated that the N-terminal helices of this domain, rather that the
hydrophobic pocket, act as a critical lipid-binding site of Tim44 (Josyula et al. 2006 ;
Marom et al. 2009 ; Cui et al. 2011 ).
Tim44 plays a dual role in the initial stages of import mediated by the PAM
complex: sensing and binding the precursor in the Tim23 channel, and recruiting
Ssc1 and Pam18/16 to the TIM23 complex (D'Silva et al. 2004 ; Marom et al. 2011 ).
Tim44 can bind Ssc1 at sites both in the ATPase and peptide-binding domains, but
does not stimulate its ATPase activity, nor interact with Ssc1 in a substrate-like
manner (Krimmer et al. 2000 ; Moro et al. 2002 ; Strub et al. 2002 ). In its ADP
bound form, Ssc1 binds to Tim44 with greater affinity than in ATP bound form
(Slutsky-Leiderman et al. 2007 ). During the protein import reaction, the complex
formed between the ADP-bound form of Ssc1 and the imported precursor dissoci-
ates from Tim44 and is released into the matrix (D'Silva et al. 2004 ). The nucleo-
tide exchange factor Mge1 then mediates subsequent dissociation of the precursor
from Ssc1, (Laloraya et al. 1995 , 1994 ; Westermann et al. 1995 ; Voos et al. 1994 ;
Schneider et al. 1996 ) with this entire cycle repeated until the precursor molecule
is imported fully into the matrix. Disruption of the N-terminal domain of Tim44 re-
duces its binding affinity for Ssc1, and affects the release of Ssc1 from Tim44 upon
substrate binding (Schiller et al. 2008 ).
Several lines of evidence point to the direct interaction of Tim44 with Pam16.
Pam16 and Tim44 can be cross-linked in vivo (Mokranjac et al. 2007 ; Kozany et al.
2004 ). Association of Pam18/Pam16 heterodimers with TIM23 complexes is sig-
nificantly reduced when Tim44, or its N-terminal region is disrupted (Schilke et al.
2012 ; D'Silva et al. 2008 ; Kozany et al. 2004 ; Hutu et al. 2008 ). Point mutations in
this region of Tim44 can suppress deleterious effects of mutations in the N-terminal
region of Pam16 (D'Silva et al. 2008 ). By interacting with the precursor proteins,
Ssc1 and the Pam18/Pam16 complex, the Tim44 co-chaperone plays a critical scaf-
folding and regulatory role in the mitochondrial protein import machinery.