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...

SummarySignal peptides target protein cargos for secretion from the bacterial cytoplasm. These signal peptides contain a tri-partite structure consisting of a central hydrophobic domain (h-domain), and two flanking polar domains. Using a recently developed in vitro transport assay, we report here that a central h-domain position (C17) of the twin arginine translocation (Tat) substrate pre-SufI is especially sensitive to amino acid hydrophobicity. The C17I mutant is transported more efficiently than wild type, whereas charged substitutions completely block transport. Transport efficiency is well-correlated with Tat translocon binding efficiency. The precursor protein also binds to non-Tat components of the membrane, presumably to the lipids. This lipid-bound precursor can be chased through the Tat translocons under conditions of high proton motive force. Thus, the non-Tat bound form of the precursor is a functional intermediate in the transport cycle. This intermediate appears to directly equilibrate with the translocon-bound form of the precursor.

Assembly of light-harvesting complexes requires synchronization of chlorophyll (Chl) biosynthesis with biogenesis of light-harvesting Chl a/b-binding proteins (LHCPs). The chloroplast signal recognition particle (cpSRP) pathway is responsible for transport of nucleus-encoded LHCPs in the stroma of the plastid and their integration into the thylakoid membranes. Correct folding and assembly of LHCPs require the incorporation of Chls, whose biosynthesis must therefore be precisely coordinated with membrane insertion of LHCPs. How the spatiotemporal coordination between the cpSRP machinery and Chl biosynthesis is achieved is poorly understood. In this work, we demonstrate a direct interaction between cpSRP43, the chaperone that mediates LHCP targeting and insertion, and glutamyl-tRNA reductase (GluTR), a rate-limiting enzyme in tetrapyrrole biosynthesis. Concurrent deficiency for cpSRP43 and the GluTR-binding protein (GBP) additively reduces GluTR levels, indicating that cpSRP43 and GBP act nonredundantly to stabilize GluTR. The substrate-binding domain of cpSRP43 binds to the N-terminal region of GluTR, which harbors aggregation-prone motifs, and the chaperone activity of cpSRP43 efficiently prevents aggregation of these regions. Our work thus reveals a function of cpSRP43 in Chl biosynthesis and suggests a striking mechanism for posttranslational coordination of LHCP insertion with Chl biosynthesis.

An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.

Background: A novel chaperone, cpSRP43, disassembles a family of membrane protein aggregates. Results: cpSRP43-mediated disaggregation requires two steps, recognition and remodeling, each with distinct molecular requirements. Conclusion: cpSRP43 uses distinct substrate binding interactions to recognize and then remodel and disrupt the protein aggregate. Significance: Mechanism of this novel ATP-independent disaggregase guides the understanding of analogous systems and design efforts to target protein aggregates of interest.