Date of Graduation

5-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Henry, Ralph L.

Committee Member

Ivey, D. Mack

Second Committee Member

Koeppe, Roger E. II

Third Committee Member

Millett, Francis S.

Keywords

Chloroplasts; Membrane binding; Protein targeting; Signal recognition particles

Abstract

A novel signal recognition particle (SRP) found in the chloroplast (cpSRP) works in combination with the cpSRP receptor, cpFtsY, to facilitate the post-translational targeting of a family of nuclear-encoded thylakoid proteins to the Alb3 translocase in thylakoid membranes. Work here focused on understanding events at the membrane that take place to ensure targeting of the cpSRP-dependent substrate to Alb3. Specifically, we sought to understand the structural and functional role of membrane binding by cpFtsY, a protein that exhibits the ability to partition between the membrane (thylakoid) and soluble (stroma) phase during protein targeting. We also sought to understand whether a novel SRP subunit (cpSRP43) in chloroplasts is involved in targeting events at the membrane beyond its role in substrate binding. Lastly, we chose to examine the possible association of Alb3 with chlorophyll (Chl) biosynthetic enzymes, which provide Chl ligands to SRP-targeted protein substrates. Our data show that cpFtsY houses a membrane-binding motif whose activity is linked to the SRP GTPase cycle. This membrane-binding motif is necessary and sufficient for binding thylakoid membranes and appears to be conserved among prokaryotic and organellar FtsY homologues. Interestingly, the removal or mutation of key residues in this region of cpFtsY results in a higher basal rate of GTP hydrolysis in solution. Furthermore, these changes correspond to a loss of lipid-induced hydrolysis stimulation, suggesting that the membrane binding region houses a negative regulator of hydrolysis is naturally switched off by a membrane-induced conformational shift. Using recombinant cpSRP43 and a construct corresponding to the soluble C-terminal extension of Alb3 (Alb3-Cterm), we show that cpSRP43 contributes to the specificity for the targeting reaction by interacting with the C-terminal region of Alb3. Furthermore, a peptide corresponding to the C-terminal region of Alb3 stimulates cpSRP GTP hydrolysis only in the presence of cpSRP43. These results suggest that cpSRP43 mediates key targeting events at the thylakoid membrane, such as release of the targeting complex from Alb3. Furthermore, these data support a model in which cpSRP43 functions as a translocon `sensing' component critical for membrane-associated steps in the post-translational cpSRP-dependent targeting pathway. Lastly, our results suggest that Alb3-dependent LHCP insertion is linked to the final stages of Chl biosynthesis. Indeed, we have identified two pools of Alb3: one that is associated with SRP targeting components and one that is associated with a late-stage chlorophyll biosynthesis enzyme (geranylgeranyl reductase). This data provides the first evidence that Chl biosynthesis enzymes are in complex with Alb3, supporting the hypothesis that the final stages of Chl biosynthesis are coordinated with the assembly of proteins that require Alb3 for assembly.

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