Mechanisms of Golgi Apparatus Assembly

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The Golgi apparatus is central to the biosynthetic processing of secretory and membrane proteins in eukaryotic cells. Proteins destined for secretion or for insertion into the plasma membrane, as well as proteins destined for various intracellular compartments such as the lysosomes, are rapidly sequestered, usually co-translationally, into an intracellular system of compartments that facilitate the post-translational biochemical processing of these proteins and their translocation to their functional locations in the cell. The first compartment into which such proteins are sequestered is the endoplasmic reticulum (ER); from there, the proteins are transported through the Golgi apparatus and from there distributed to their ultimate locations. The Golgi apparatus itself is complex, and its various components (stacked cisternae) are specialized for various functions, e.g., in terms of postranslational modifications of proteins. The mechanism of transport of proteins through the various cisternae of the Golgi apparatus, and the biogenesis and maintenance of the Golgi apparatus in the cell, is a topic of considerable significance to the understanding (and, ultimately, deliberate biotechnological manipulation) of these processes. It is also a controversial topic, since recent discoveries have forced cell biologists to reconsider the working model of Golgi biogenesis that has dominated cell biological thinking in this area for the past twenty years. This proposal addresses a key aspect of this issue, the functional and dynamic relationship of the Golgi apparatus to the endoplasmic reticulum.The Golgi apparatus receives newly synthesized proteins and lipids from the endoplasmic reticulum (ER) and distributes them to other downstream organelles within the secretory pathway. Key to Golgi function is an unequal distribution of 'resident' proteins, that are concentrated in one cisterna and are absent from other parts of the organelle, to give a 'polarized' stack of Golgi membranes. The proposed research centers on the question of how polarized Golgi stacks form; in other words, on the biogenesis of the organelle. Prior work by Dr. Storrie (Storrie et al., 1998; Storrie and Yang, 1998) raises the possibility that the Golgi apparatus may 'melt' into the ER during mitosis followed by subsequent de novo formation of the organelle from the ER. In this project, exploratory research will be performed to test the role of two families of molecules in the postulated de novo formation of polarized Golgi stacks. These molecules are the COPI class of coat proteins and two members of the rab family of small GTPases, rab6 and rab33b. Each is known from the work of others to be involved in membrane trafficking. Dr. Storrie will assess conditions that specifically block vesicular transport within the Golgi stack and will score for effects on de novo Golgi formation using as a model system Golgi reassembly from the ER following brefeldin A (BFA) washout. BFA-treatment results in a reversible dispersal of Golgi proteins and lipids to the ER. The experiments probe the hypothesis that vesicular transport processes may be necessary to mold stabilized molecular associations within the Golgi apparatus into polarized Golgi stacks. A reverse genetic approach will be taken, in which dominant negative mutant proteins will be introduced into cells by microinjection.