PARENT SESSION
Posters P7C Biosynthesis and assembly: Protein trafficking. Abstracts (681-684)

Analysis of oligomeric interactions and GTP binding/hydrolysis activities of the soluble domain of the Toc GTPases. Barry Bruce^*,1, Sarah Wright¹, Evan Reddick¹, Michael Vaughn¹, Kenny Minor¹, ¹ Biochemistry, Cellular, & Moelcular Biology Department, knoxville, TN, USA

ABSTRACT- The majority of the plastid-localized proteins are nuclear-encoded and post-translationally imported from the cytosol. The mechanism for the selective targeting and translocation of the precursor appears to be through recognition of the transit peptide by the Toc GTPases, which act as receptors or gate-keepers. Unfortunately, very little is known about the structure and dynamics of the Toc apparatus. Recent work has demonstrated a complex interaction between the Toc GTPases that is modulated by preproteins/transit peptides and GTP/GDP. To aid in the elucidation of this complex process, we have developed highly sensitive GTP-binding and hydrolysis assays to characterize the soluble domain of four Toc GTPases: atToc33, atToc34, psToc34 and atToc159G. We have determined the Vmax and Km for these GTPases. We have also 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 also able to distinguish between a stimulation of GTP hydrolysis vs. stimulation of nucleotide exchange. We have determined the Kd/Ki for a wide range of nucleotides. We are also exploring the possibility of trans stimulation of the Toc GTPases via a reciprocal GEF or GAP activity. Evidence for direct Toc-Toc interactions can be seen from the psToc34 crystal structure, and the low-resolution structure of the isolated Toc complex. We have enlisted analytical ultracentrifugation to measure the interactions between the soluble domains of atToc33, atToc34, atToc159G, & psToc34. Using velocity centrifugation we have observed the presence of a homodimer of psToc34, atToc33, and atToc34. Interestingly, the population of this homodimer is influenced by the addition of the full-length transit peptide, SS-tp. We are extending this analysis to determine how the oligomeric state of the Toc subunit is effected by a) addition of hydrolyzable and non-hydrolyzable nucleotides, b) addition of a second Toc GTPase (for example, atToc159G), c) effect of site-directed mutations of SS-tp. 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.

KEY WORDS: translocon, GTPase, transit peptide, protein import