Membrane protein biogenesis poses enormous challenges to cellular protein homeostasis and requires effective molecular chaperones. Compared with chaperones that promote soluble protein folding, membrane protein chaperones require tight spatiotemporal coordination of their substrate binding and release cycles. Here we define the chaperone cycle for cpSRP43, which protects the largest family of membrane proteins, the light harvesting chlorophyll a/b-binding proteins (LHCPs), during their delivery. Biochemical and NMR analyses demonstrate that cpSRP43 samples three distinct conformations. The stromal factor cpSRP54 drives cpSRP43 to the active state, allowing it to tightly bind substrate in the aqueous compartment. Bidentate interactions with the Alb3 translocase drive cpSRP43 to a partially inactive state, triggering selective release of LHCP's transmembrane domains in a productive unloading complex at the membrane. Our work demonstrates how the intrinsic conformational dynamics of a chaperone enables spatially coordinated substrate capture and release, which may be general to other ATP-independent chaperone systems.membrane protein biogenesis | molecular chaperone | signal recognition particle | protein dynamics | NMR spectroscopy P rotein homeostasis is essential for all cells and requires proper control of the folding, localization, and interactions of proteins. The biogenesis of membrane proteins poses a particular challenge to protein homeostasis. Before arrival at the membrane, newly synthesized membrane proteins need to traverse aqueous cellular compartments where they are highly prone to aggregation. Thus, the posttranslational targeting of membrane proteins relies critically on effective molecular chaperones that maintain nascent membrane proteins in translocation competent states. Many examples illustrate the intimate link between chaperone function and membrane protein biogenesis: SecB, Skp, and SurA protect bacterial outer membrane proteins (1-5), and Hsp70 homologs assist the import of mitochondrial or chloroplast proteins (6).Our understanding of membrane protein chaperones lags far behind that for soluble proteins, such as DnaK and GroEL. All chaperones need to switch between "open" and "closed" conformations to allow substrate release and binding, respectively. For many chaperones that promote the folding of soluble proteins, these switches can be driven either by ATPase cycles, such as Hsp70 (7) and GroEL (8), or by changes in environmental conditions, such as the acid-induced HdeA (9, 10) and oxidationinduced Hsp33 (11). In contrast, membrane protein chaperones must regulate their action spatially: they must effectively capture substrate proteins in the aqueous phase, and then facilely and productively release them at the target membrane. With few exceptions (1, 2), how membrane protein chaperones achieve spatiotemporal coordination of their chaperone cycle is not well understood.The light harvesting chlorophyll a/b-binding proteins (LHCPs) provide an excellent model system to address these quest...

A method for the Pd-catalyzed N-arylation of both aryl and alkyl amidines with a wide range of aryl bromides, chlorides, and triflates is described. The reactions proceed in short reaction times and with excellent selectivity for monoarylation. A one-pot synthesis of quinazoline derivatives, via addition of an aldehyde to the crude reaction mixture following Pd-catalyzed N-arylation, is also demonstrated.

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, ...

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The chloroplast signal recognition particle 43 (cpSRP43) is an ATP‐independent chaperone that delivers the light‐harvesting chlorophyll‐binding proteins (LHCPs) to the thylakoid membrane while also maintaining the levels of tetrapyrrole biosynthesis (TBS) proteins. How cpSRP43 coordinates these roles to protect two very different classes of clients has remained unclear. We show that cpSRP43 samples two distinct conformations: a well‐folded closed state that protects unfolded membrane protein LHCPs through contacts with its substrate binding domain (SBD) and an open state with a partially disordered SBD that is less active towards LHCP but remains effective at protecting TBS proteins from heat‐induced aggregation. Binding of the additional cpSRP54 subunit stimulates cpSRP43's chaperone activity towards LHCPs by stabilizing the closed state but inhibits its chaperone activity towards TBS proteins. Strikingly, the cpSRP43‐cpSRP54 binding affinity is greatly weakened at high temperatures. Taken together, this shows that temperature can direct cpSRP43 towards one of these two client pathways. Under normal conditions, most cpSRP43 is tightly bound to cpSRP54 in the closed state and is primarily dedicated to the protection and delivery of LHCPs. However during heat stress, as chloroplast proteins become increasingly susceptible to heat‐induced aggregation, cpSRP43 dissociates from cpSRP54 and is rapidly repurposed into a stress‐responsive chaperone for TBS proteins.