Protein translocation across biological membranes is required for the proper localization of over half the proteins of a typical cell. The chloroplast twin-arginine translocation (cpTat) pathway transports protein from the stroma of the chloroplast into the thylakoid lumen. Substrates on the cpTat pathway interact with Hcf106-TatC receptors, and are translocated by the assembly of Tha4 in the presence of a trans-thylakoidal protonmotive force, which also provides the driving force for protein translocation. PspA, the bacterial homolog of a chloroplast protein, Vesicle-Inducing Protein in Plastids 1 (VIPP1), has been shown to stimulate Tat transport in E. coli.
This dissertation sets out to investigate whether VIPP1 plays a role in cpTat transport in the chloroplast. VIPP1 is localized to the chloroplast inner envelope membrane, stroma, and thylakoid membrane. It has been shown to be required for proper thylakoid biogenesis and organization. Recently, a number of studies have suggested VIPP1 activity may not be restricted to membrane vesicle formation.
This work demonstrates that VIPP1 protein stimulates cpTat transport activity in in vitro thylakoid transport assays. VIPP1 increases cpTat transport by enhancing the substrate-binding step in the cpTat pathway. Thylakoids supplemented with VIPP1 bind up to twice the amount of substrates compared to untreated thylakoids, which results in increased cpTat transport. This is in contrast to the mechanism proposed for PspA stimulation of Tat transport in bacteria; via recovery of the electric-chemical potential of leaky membranes. The amount of substrate bound onto the thylakoid with VIPP1 supplementation exceeds the predicted number of cpTat receptor sites, suggesting substrates interact directly with the thylakoid membrane. This supports the hypothesis that protein substrates may bind the membrane directly as an early step in the cpTat pathway, as have been shown for Tat transport in bacteria.
VIPP1 also showed an ability to increase sedimentation of thylakoid preparations. Thylakoids are organized into grana stacks and stromal lamallae, with cpTat components found at the stroma-exposed regions of the thylakoid membrane. This work may be used for development of further hypothesis involving the role of VIPP1 on the organization of the thylakoid membrane and the consequences to cpTat transport.
|Advisor:||Theg, Steven M.|
|Commitee:||Harada, John J., Inoue, Kentaro|
|School:||University of California, Davis|
|School Location:||United States -- California|
|Source:||DAI-B 73/07(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Plant biology|
|Keywords:||Chloroplast, Cptat, Protein transport, Thylakoid, Vipp1|
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