The general goal of these studies is to further our understanding of the functional organization and the mechanism of assembly of the plant Golgi apparatus. The research will take advantage of unique features of plant Golgi stacks such as their structural stability to address some of the more intractable problems in Golgi research in general: how the stacked architecture is created and maintained, and how Golgi stacks are retailored during cell differentiation. In addition, we propose to determine how their mobility changes during the cell cycle and during development. To study cell type specific retailoring of Golgi stacks, we will exploit a novel model system of plant cell differentiation that we have developed. By depriving tobacco BY-2 cell cultures of the auxin hormone 2,4-D, these cells can be induced to differentiate in a manner that resembles the differentiation of slime-secreting cells in root tips, importantly including the remodeling of their Golgi stacks. Our principle aim is to relate the changes in Golgi architecture to the underlying changes in the protein composition of the membranes. Selected proteins that are either up- or down-regulated will be purified for microsequencing and for producing antibodies with the goal of localizing the proteins and cloning the genes. The second line of work will build on our discovery of a method for producing artificial organelles called Z-(zippered) membranes that can be used for overexpressing membrane proteins in partly sequestered membrane domains of the ER. Here we propose to produce LZ-(lumenally zippered) membranes to overexpress Golgi proteins in discrete sheet-like structures that are continuous with the ER. These artificial organelles will be used to identify membrane proteins that interact with overexpressed Golgi proteins to test current Golgi protein retention theories. The same LZ- membranes will be used as affinity matrices for identifying cytosolic proteins that can bind to N-terminal domains of Golgi proteins and thereby potentially mediate membrane stacking. Alternative approaches for identifying such proteins such as using a lambda expression library or an affinity chromatography system are also discussed. Finally, we propose to use an alpha-mannosidase-I-green fluorescence protein fusion protein to investigate Golgi dynamics and test the hypothesis that plant Golgi stacks move around during cytoplasmic streaming, as well as to see in transgenic plants how the dynamics of Golgi stacks changes during tissue differentiation.
