Poster: Protein targeting & vesicular trafficking

Abs # 654: Modulation of GTP-binding and hydrolysis activity of recombinant forms of the chloroplast Toc GTPases

Presenter:	Reddick, Lovett E, lreddick@utk.edu
Authors	Reddick, Lovett E (A)   Vaughn, Michael D (A)   Wright, Sarah J (A)   Bruce, Barry D (A)
Affiliations:	(A): Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996

The chloroplast must import the majority of its proteins as nuclear-encoded larger molecular weight precursors. Import of these precursors is mediated via sequential interactions of the transit peptide and one or more components of the Toc apparatus (Translocon at the Outer membrane of the Chloroplast). Recent work has demonstrated a complex interaction between the Toc GTPases and their substrates: preproteins/transit peptides and nucleotides (GTP/GDP). To simplify and aid in the elucidation of this complex process, we have developed highly sensitive GTP-binding and hydrolysis assays. Using these assays we have characterized the soluble (ΔTM) domain of four different Toc GTPases: atToc33, atToc34, psToc34 and atToc159G. We have determined the Vmax (650-1328 fmol GTP hydrolyzed/min/pmol Toc) and Km (48-140 μM) for these four related GTPases. Using these enzymatic parameters, we have found that transit peptides and precursor proteins stimulate the GTPase activities, while the mature proteins either do not stimulate or suppress activity. Using an immuno/affinity magnetic recovery method we are now able to distinguish between a stimulation of GTP hydrolysis vs. stimulation of nucleotide exchange. We have also observed that the rate of GTP hydrolysis is greatly stimulated by the addition of chloroplast-mimetic liposomes as well as select detergent micelles. We are also using combinations of the atToc GTPases to explore the possibility of trans stimulation via reciprocal GEF or GAP activity. Finally, the use of molecular modeling has allowed the design of site-specific mutations that will yield mutated GTPases with altered substrate specificity, dimerization properties and preprotein recognition.