Poster: Organelle biogenesis

Abs # 633: Imaging and cytometric analysis of the macromolecular organization and assembly of Toc components in intact plastids.

Presenter:	Mitsky, Anton V, amitsky@utk.edu
Authors	Mitsky, Anton V (A)   Subramanian, Chitra  (A)   Joy, David  (A)   Allison, David  (A)   Bruce, Barry  (A)
Affiliations:	(A): University of Tennessee at Knoxville

It is been estimated that in a typical plant more than 3000 gene products are directed to the plastid via the transit peptide-mediated pathway of post-translational translocation. Recently, considerable progress has been made to identify the components of Toc (Translocator of the outer chloroplast membrane) and Tic (Translocator of the inner chloroplast membrane) apparatus. However, the details of how these components are organized is poorly understood and has only partially been described via a low resolution structure of an isolated Toc complex using a combination of electron microscopy and image analysis. This purified complex(s) consisted of only three proteins: Toc 34, Toc 75 and Toc 159 in 4:4:1 ratio, respectively. However, this imaged complex represents only one subset of 8 resolved particle classes, nor did this study address the organization of several smaller Toc complexes with different subunit composition. Because of the difficulties and potential artifacts associated with the analysis of detergent solubilized Toc complex(s), we are exploring the use of novel microscopic and flow cytometric analyses of the Toc complex in situ using isolated plastids and envelopes. Our group employs immunocytochemistry in conjunction with such techniques as TEM, VP-SEM, LSCM, AFM and flow cytometry. Using these methods, we attempt to monitor the assembly, organization and dynamics of the Toc complex as a function of developmental age, plastid subtype, transit peptide structure and NTP availability.