ER-Bound Steps in the Biosynthesis of G Protein-Coupled Receptors - GPCR Signalling Complexes: Synthesis, Assembly, Trafficking and Specificity - page 16

common a zinc finger-like domain thought to mediate client recognition. The
J-domain provides the physical link to HSP70 proteins and is necessary to enhance
the intrinsic HSP70 ATPase activity. ADP-bound HSP70 tightly associates with the
client protein and the association releases on exchange of ADP for ATP (Bermak
et al. 2001 ; Qiu et al. 2006 ). However, HSP40 proteins need not cooperate with
HSP70 partner for individual functions (Vembar et al. 2009 ). A few receptors have
been reported to associate with cytoplasmic (overexpressed) HSP40 proteins and
this reduces receptor export and surface expression (Bermak et al. 2001 ; Chapple
and Cheetham 2003 ; Málaga-Diéguez et al. 2010 ; Meimaridou et al. 2011 ) . However,
there is little evidence to substantiate the assumption that HSP40 proteins bind to
the proximal carboxyl terminus, i.e ., the helix VIII sequence of the receptor. They
may rather recognize a deficiency in packing of the transmembrane domain when
hydrophobic peptide sequences protrude from the lipid bilayer.
Experimental evidence is limited for additional components that could ascertain
the folding status of the transmembrane domain. One candidate is the polytopic
ER membrane protein HRD1, which has been discovered because it disposes of
yeast Hmg2p. Hmg2p is the yeast isoform of HMG-CoA (hydroxymethylglutaryl
coenzyme A) reductase, an ER-resident transmembrane protein. In yeast, HRD1
consists of an N-terminal membrane anchor linked to a soluble C-terminal domain
with a RING-H2 domain characteristic of many E3 ubiquitin ligases. HRD1 is
required for the degradation of ERAD-L and ERAD-M substrates and cooper-
vates with valoesin-containing protein p97, an ATPase that provides the force to
extract substrates from the ER (Brodsky and Skach 2011 ). For the recognition of
luminal substrates HRD1 relies on partners (lectins or classical chaperones)
whereas ERAD-M substrate recognition was proposed to represent an intrinsic
feature of the protein (Sato et al. 2009 ). Ubiquitin ligation is considered the rate-
limiting step in HRD1-mediated degradation. The C-terminal region of HRD1 is
responsible for catalyzing the transfer of ubiquitin to ERAD substrates for which
it requires the presence of the N-terminal membrane anchor. The multi-spanning
HRD1 thus has several functions: these encompass - at the very least -recognition
and ubiquitination of retained membrane proteins and turning them into ERAD
substrates.
In line with the organization of ERAD-M in yeast, the extracting ATPase p97
was reported to enhance the turnover of retained rhodopsin (Griciuc et al. 2010 ) .
Similarly, HRD1 triggers degradation of mutated opsin in mammalian cells, if two
hydrophilic basic residues are introduced in a-helix I as a degradation-inducing
motif (“degron”) (Ray-Sinha et al. 2009 ); the substitution of hydrophobic residues
normally buried in the lipid environment of the bilayer suffices for recognition by
ERAD-M. Like in yeast HMG2p, detection of the inappropriate residues may be
accomplished by direct interaction with the membrane-embedded hydrophilic
residues on the HRD1 transmembrane domain. The evidence for an analogous
mechanism is not definitive. However, the observations support the conjecture that
defects at the level of the transmembrane domain can be probed by a transmem-
brane chaperone and ERAD component.