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...