Two distinct sites of client protein interaction with the chaperone cpSRP43

Camille Z. McAvoy, Alex Siegel, Samantha Piszkiewicz, Emily Miaou, Mansen Yu, Thang Nguyen, Annie Moradian, Michael J. Sweredoski, Sonja Hess, Shu-ou Shan
2018 Journal of Biological Chemistry  
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
more » ... served 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, and that it is necessary and sufficient to chaperone LHCPs (21, 22) . Although the chaperone activity of cpSRP43 is allosterically regulated by additional components in the cpSRP pathway, such as cpSRP54 and Alb3 (19, (22) (23) (24) , the simple composition of the cpSRP43-LHCP chaperone-client pair and the robustness of cpSRP43's chaperone activity make this pair an excellent system to understand the interaction and regulation of membrane protein chaperones. A long-standing question about the cpSRP43 chaperone is the mechanism by which it prevents the hydrophobic TMDs on its substrate proteins from aggregation. The substrate-binding domain (SBD) of cpSRP43 is comprised of ankyrin repeat motifs 1-4, capped at the N-terminus by a chromodomain (CD1) and at the C-terminus by a bridging helix (BH) (21, 22, 25) . Biochemical and crystallographic analyses showed that a conserved Tyr204 in the third ankyrin repeat motif recognizes an FDPLGL motif in L18, a conserved 18-amino acid sequence between the second and third TMDs of LHCP (12, 22, (25) (26) (27) . However, interaction with a soluble loop sequence is unlikely to be sufficient to protect LHCPs, which contain three TMDs, from aggregation. The ability of cpSRP43 to quantitatively prevent full-length LHCPs from aggregation is highly suggestive of additional interactions between cpSRP43 and the substrate TMDs. Moreover, a recent study showed that cpSRP43 also protects aggregation-prone regions of glutamyl-tRNA reductase to enhance the stability of this enzyme (28), indicating that cpSRP43 can contact hydrophobic segments on client proteins independently of L18 recognition. Nevertheless, deletion of individual TMDs in LHCP or replacement with the TMDs from unrelated membrane proteins did not severely disrupt the cpSRP43-LHCP interaction
doi:10.1074/jbc.ra118.002215 pmid:29669809 fatcat:iqtbfgb6irbsbp2xjo4lir4dai