Abstract
The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane.
Highlights
Proper compartmentalization of proteins relies on the ability of protein localization pathways to transport proteins effi
Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpFtsY functions along with a unique SRP (cpSRP)-dependent protein targeting
Lightharvesting chlorophyll a/b-binding proteins (LHCPs) imported into the chloroplast stroma are bound by cpSRP to form a soluble targeting complex, which directs the LHCP substrate to the thylakoid membrane translocon Alb3 (Albino3) in a GTP- and cpFtsY-dependent manner [14, 36]
Summary
All reagents and enzymes used were purchased commercially. All primers were from Integrated DNA Technologies. The cpSRP43 transcription/translation clone was designed to match the mature coding sequence of A. thaliana cpSRP43 starting with Ala-Ala-ValGln-Arg-Asn and incorporating a Kozak sequence and restriction sites for insertion into pGEM-7Z. Protein Integration Assays Using Isolated Salt-washed Thylakoids—Integration assays included salt-washed thylakoids (equal to 50 g of chlorophyll) in IBM buffer, 5 mM ATP, 14892 JOURNAL OF BIOLOGICAL CHEMISTRY. Assays for Determining Membrane Binding and Partitioning— Partitioning assays included thylakoids (equal to 75 g of chlorophyll) in IBM buffer and radiolabeled translation products. CpFtsY Membrane Binding Saturation Curves—Salt-washed or protease-treated thylakoids (equal to 50 g of chlorophyll) were incubated with 0, 1, 2, 4, 8, 16, 32, or 64 g of cpFtsY in a final volume of 100 l of IBM buffer. Thylakoids were re-isolated, washed, and resuspended to a final volume of 50 l, and 5 l of each sample was analyzed by SDS-PAGE. The brominated lipid mixture was extruded through 80-nm polycarbonate membranes and homogenized via freeze/thaw cycles
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