Two distinct sites of client protein interaction with the chaperone cpSRP43

Abstract

Integral membrane proteins are prone to aggregation and misfolding in aqueous environments and therefore require binding by molecular chaperones during their biogenesis. Chloroplast signal recognition particle 43 (cpSRP43) is an ATP-independent chaperone required for the biogenesis of the most abundant class of membrane proteins, the light-harvesting chlorophyll a/b-binding proteins (LHCPs). Previous work has shown that cpSRP43 specifically recognizes an L18 loop sequence conserved among LHCP paralogs. However, how cpSRP43 protects the transmembrane domains (TMDs) of LHCP from aggregation was unclear. In this work, alkylation-protection and sitespecific crosslinking experiments found that cpSRP43 makes extensive contacts with all the TMDs in LHCP. Site-directed mutagenesis identified a class of cpSRP43 mutants that bind tightly to the L18 sequence but are defective in chaperoning full-length LHCP. These mutations mapped to hydrophobic surfaces on or near the bridging helix and the β-hairpins lining the ankyrin repeat motifs of cpSRP43, suggesting that these regions are potential sites for interaction with the client TMDs. Our results suggest a working model for client protein interactions in this membrane protein chaperone.Proper protein folding and localization are critical for cellular protein homeostasis. The posttranslational targeting of integral membrane proteins poses an acute challenge to protein homeostasis. Before arrival at the target membrane, nascent membrane proteins are highly prone to aggregation in the cytosol and other aqueous cellular compartments. Thus, effective molecular chaperones or chaperone networks are required to minimize improper exposure of the transmembrane domains (TMDs) on newly synthesized membrane proteins and to maintain them in a soluble, translocation-competent conformation. Many examples illustrate the intimate link between chaperone function and membrane protein biogenesis, including SecB, Skp, and SurA that protect bacterial outer membrane proteins, and Hsp70 homologues implicated in the import of precursor proteins to the endoplasmic reticulum, mitochondria or chloroplast (1-7).The light-harvesting chlorophyll a/b-binding proteins (LHCP) comprise over 50% of the protein content on the thylakoid membrane of green plants and form the most abundant family of membrane proteins on earth (8). LHCPs are nuclear encoded, initially synthesized in the cytosol, and imported across the chloroplast envelope in a largely unfolded state (8). In the chloroplast stroma, LHCPs are protected in a soluble 'transit complex' by the chloroplast signal recognition particle (cpSRP), comprised of the cpSRP43 and cpSRP54 protein subunits (9-12). Via interactions between the GTPase domains of cpSRP54 and its receptor cpFtsY, LHCPs are delivered to the Alb3 translocase and inserted into the thylakoid membrane (11,(13)(14)(15)(16)(17)(18)(19)(20). Previous work showed that the cpSRP43 subunit binds tightly to and quantitatively prevents the aggregation of multiple members of the LHCP family, ...

Chloroplast SRP43 subunit Prevents Aggregation of Proteins

“…The conserved Tyrosine at position 204 in ANK3 of cpSRP43 recognizes the FDPLGL sequence in L18 (7,8,15,20,22). However, this interaction alone would not be adequate to guard the trans-membrane domains (TMDs) from aggregation (47). Investigation into how cpSRP43 can protect the TMDs of LHCP has only recently been communicated.…”

“…Investigation into how cpSRP43 can protect the TMDs of LHCP has only recently been communicated. Mapping of interaction sites between cpSRP43 and LHCb5 was done by alkylation efficiency of LHCb5 cysteine mutants using N-ethylmaleimide followed by sitespecific cross-linking and the results of this combination of experiments revealed that cpSRP43 makes contacts and protects all three TMDs of the LHCb5 substrate (47). In site-directed mutagenesis studies, residues were categorized into two classes being mutations which disrupted cpSRP43's chaperoning of LHCP but not the binding of L18 and mutations in which both were disrupted (47).…”

“…Mapping of interaction sites between cpSRP43 and LHCb5 was done by alkylation efficiency of LHCb5 cysteine mutants using N-ethylmaleimide followed by sitespecific cross-linking and the results of this combination of experiments revealed that cpSRP43 makes contacts and protects all three TMDs of the LHCb5 substrate (47). In site-directed mutagenesis studies, residues were categorized into two classes being mutations which disrupted cpSRP43's chaperoning of LHCP but not the binding of L18 and mutations in which both were disrupted (47). These experiments were the basis for designing a model of the interaction surface on cpSRP43 for the protection of the TMDs of LHCP which is comprised of hydrophobic surfaces on ANK4, the bridging helix at the N-terminal of the ankyrin regions and, the beta-hairpins along the ankyrin regions (47).…”

“…In site-directed mutagenesis studies, residues were categorized into two classes being mutations which disrupted cpSRP43's chaperoning of LHCP but not the binding of L18 and mutations in which both were disrupted (47). These experiments were the basis for designing a model of the interaction surface on cpSRP43 for the protection of the TMDs of LHCP which is comprised of hydrophobic surfaces on ANK4, the bridging helix at the N-terminal of the ankyrin regions and, the beta-hairpins along the ankyrin regions (47). Since cpSRP43 is a known chaperone of LHCPs, it is conceivable that the binding interface between these two proteins can be elucidated in detail upon adequate investigation.…”

“…We have already seen that cpSRP43 undergoes differing conformational rearrangements upon binding to partners along the cpSRP pathway (52). In addition, the exceptional ability of cpSRP43 to solubilize a substrate is key for its chaperoning function and can be achieved through contacts being hydrophobic and electrostatic in essence (1,4,6,8,47,53,54). These exceptional properties of cpSRP43 may make it challenging to define the binding interface between cpSRP43 and other substrates outside of the cpSRP system.…”