Protein Targeting and Vesicular Trafficking
Reddick, L. Evan , Wright, Sarah J. , Chotewutmontri, Prakitchai , Wright, Stephen , Bruce, Barry D. .
Modulation of Toc GTPase Activity and Conformation by Substrate, Homotypic and Heterotypic Interactions.
Unique to protein transport into chloroplasts is the involvement of the receptor GTPases Toc34 and Toc159 that are found in the translocon of the outer envelope (Toc). Although these proteins have been proposed to function as receptors and/or gatekeepers, very little is known about their structure and dynamics. Their role is complex due apparent interactions with preprotein/transit peptide and nucleotide (GDP/GTP), as well as their ability to homo- and heterodimerize. We have recently reported a careful characterization of the enzymatic properties of these proteins as well as the ability of preproteins and transit peptides to stimulate their hydrolytic rates. Both prSSU and its full-length transit peptide (SStp) have been shown to stimulate the rate of GTP hydrolysis. Interestingly, the full-length preprotein has a greater activity that the full-length transit peptide, supporting earlier work that the mature domain somehow modulates the activity; the transit peptide does not increase the rate of nucleotide exchange thus indicating that it acts as a GAP, or GTPase Activating Protein. Extending this study, we explore how these full-length transit peptides (SStpPs and SStpNt) influence the oligomeric nature of the cytosolic domain of the Toc GTPases using AUC. In addition, we are exploring how non-hydrolysable and transition state GTP analogs influence the oligomeric state of psToc34. Finally, using both fluorescent GTP-analogs and environmental-sensitive fluorescent reporters, we are probing conformational changes that occur during transit peptide:psToc34 interaction. By combining mutagenesis with both conformational and enzymatic data, we have developed a model of how transit peptide/Toc protein interactions modulate the GTPase cycle during protein import.
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1 - University of Tennessee, Biochemistry and Cellular and Molecular Biology, 224 Hesler Biology Building, Knoxville, TN, 37996, USA
2 - University of Tennessee, Biochemistry and Cellular and Molecular Biology
3 - University of Tennessee, Graduate School of Genome Science and Technology
4 - Carson-Newman College, Biochemistry
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM