UCS Proteins: Chaperones for Myosin and Co-Chaperones for Hsp90 - The Networking of Chaperones by Co-chaperones - page 135

active form in C2C12 mouse cell line suggesting that this myosin requires addition-
al cofactor(s) which are present in myogenic cells for folding (Kinose 1996 ; Chow
et al. 2002 ). In vitro , chimeric fast skeletal muscle myosin head fused to green fluo-
rescent protein (GFP) folds very slowly and transits through multiple intermediates
in a temperature-dependent manner that strongly suggests a high susceptibility to
off-pathway interactions and aggregations and hence the need for chaperone-assist-
ed folding (Chow et al. 2002 ). Expression of the protein in vivo is cell-dependent:
C2C12 myogenic cell lines yield a folded and active protein that exhibits Mg 2+
ATP-sensitive actin-binding and myosin motor activity, while epithelia cell lines
yield inactive protein aggregates (Chow et al. 2002 ). This observation suggests that
the myosin motor requires cytosolic molecular chaperones to fold correctly under
physiological conditions and that the required factor(s) are optimized in muscle
cells (Chow et al. 2002 ). In addition, during de novo folding and assembly of stri-
ated muscle myosin heavy chain, Hsp70 and Hsp90 colocalize with the myosin
intermediates but not the mature myofibrils (Srikakulam and Winkelmann 2004 ).
Using biochemical analyses, Liu et al. ( 2008 ) and Srikakulam and Winkelmann
( 2008 ) showed that UNC-45 forms a stable complex with Hsp90 that specifically
binds unfolded myosin motor and promotes its folding.
UCS Proteins-Hsp90 Interaction in Myosin Folding,
Assembly and Function
Genetic, biochemical and structural studies have confirmed that UCS proteins, es-
pecially UNC-45 interact with Hsp90 chaperone (Barral et al. 2002 ; Mishra et al.
2005 ; Srikakulam and Winkelmann 2004 , 2008 ; Liu et al. 2008 ; Gazda et al. 2013 ).
Full-length UNC-45 from C. elegans binds both endogenous Hsp70 and Hsp90
from Sf9 insect cell lysates (Barral et al. 2002 ). Mutant UNC-45 protein lacking the
TPR domain interacts with Hsp70 but not Hsp90 also from Sf9 insect cell lysates,
indicating that the interaction of UNC-45 with Hsp90 is specifically mediated by
the TPR domain (Barral et al. 2002 ). In surface plasmon resonance spectroscopy
experiments, the binding of Hsp90 to the TPR domain of UNC-45 is preferentially
competed by Hsp90 C-terminal peptides in comparison to the analogous Hsp70
peptides (Barral et al. 2002 ). C-terminal peptides of both Hsp70 and Hsp90 have
been co-crystallized with the TPR domain of UNC-45 (Gazda et al. 2013 ). Purified
Hsp90, myosin and UNC-45 can form the three possible binary complexes in pull-
down assays (Barral et al. 2002 ). When expressed in striated muscle cells, UNC-
45 was isolated as a stable complex with Hsp90 (Liu et al. 2008 ; Srikakulam and
Winkelmann 2008 ). Both the myosin binding and chaperoning activities of UCS
proteins have been mapped to the UCS domain of the protein (Barral et al. 2002 ;
Mishra et al. 2005 ; Srikakulam and Winkelmann 2004 , 2008 ; Liu et al. 2008 ; Shi
and Blobel 2010 ; Ni et al. 2011 ). Therefore both UNC-45 and Hsp90 (Du et al.
2008 ; Gaiser et al. 2011 ) are capable of interacting directly with and exerting chap-
eroning activity on myosin motor domain and thick filament assembly. However,