The past three years have witnessed a major paradigm shift in Golgi trafficking research away from models which postulate that transport through Golgi sticks is mediated by vesicles to models that support a cisternal progression/maturational theory of Golgi trafficking. Central to these latter models is the formation of new cisternae on the cis-side of the stacks, the maturation of cisternae as they are displaced across the stacks, and the sorting and packing of products and the detachment and fragmentation of cisternae on the trans-side of the stacks. To learn more about these processes we propose to use a combination of advanced structural research techniques (cryofixation, freeze-substitution and dual- axis tomography) to produce high resolution (approximately 6 nm) 3-D models of the Golgi apparatus of selected models systems. The three model systems to be investigated are: (1) the yeast Pichia pastoris, which produces Golgi stacks and is particularly suited for investigating the relationship between transitional ER (tER) sites and Golgi stack assembly; (2) sale-forming algae, the """"""""classical"""""""" systems for Golgi cisternal progression research; and (3) two plant cell types that produce different types of secretory products. The reasons for choosing these Golgi model systems are their small size and greater simplicity compared to mammalian Golgi, and strong experimental evidence that they do operate according to the cisternal progression/maturation models. In addition, each system offers unique experimental advantages such as genetic tractability (P. Pastoris), the ability to alter the types of products produced and the mode of operation of the Golgi stacks without drug treatments (scale-forming algae), the possibility of monitoring the state of assembly of specific Golgi products in different cisternae by morphological means (scale-forming algae), and the possibility of characterizing a Golgi system that appears to operate without a trans Golgi network (TGN, plant cells). Taken together, these comparative studies should help define structures that are common to all Golgi and thereby put novel morphological constraints on models of Golgi function.
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