Matrix Proteins of the plant ER-Golgi interface

The Golgi apparatus is a key component within cells which is involved in the processing and manufacture of proteins, carbohydrates and membranes which are targeted to other compartments of the cells of secreted to the external environment. We have previously shown that in plant cells the Golgi apparatus is unique consisting of numerous stacks of membrane-bounded discs called cisternae distributed within the cell. In many cell types these Golgi stacks are also very closely associated with another membrane network, the endoplasmic reticulum that passes biosynthetic cargo to the Golgi for processing. One remarkable feature of the plant Golgi, which distinguishes it from that in animal and fungal cells, is that the stacks are highly motile and in many tissues including leaves, move over the surface of the endoplasmic reticulum. The key questions that we are going to address in this research programme are how does the Golgi stay attached to the ER and how do the individual stacks of cisternae remain intact when they are moving through the cytoplasm? Over recent years a number of proteins have been discovered that may potentially act as anchors or tethers that help hold the Golgi together and which may also act as linkers for transport vesicles produced by the endoplasmic reticulum. We have recently identified a number of proteins that are good candidates for the plant tethering proteins including one novel protein. Using the technology of fusing these to fluorescent marker proteins we have shown these to locate to the part of the Golgi nearest to the endoplasmic reticulum. The work proposed in this grant will involve investigating the interaction of these proteins with each other and the regulation of these proteins. Their role in organising the Golgi stack will be assessed by expressing in leaves mutant forms of the proteins and looking for both morphological and biochemical effects on the Golgi apparatus and the secretion of proteins. We will also investigate the role these proteins may have in the biogenesis of new Golgi stacks from the endoplasmic reticulum.

As part of an existing grant (joint SEERAD/BBSRC funding with the group of K. Oparka, SCRI) we have cloned a number of proteins which are putative tethering complexes or matrix proteins predicted to be associated with the plant Golgi apparatus. These include a number of coiled coil proteins termed Golgins including homologues of mammalian golgin-84, CASP and p115. We have also isolated a novel coiled-coil CASP binding protein which appears to be unique to plants. All of these proteins when expressed in tobacco leaves as fluorescent protein fusions locate to the Golgi apparatus. In this proposal we intend to confirm the ultrastructural location of these proteins by immunocytochemistry of both native proteins and fluorescent protein expressed constructs, and to establish in vivo the interactions between these proteins and between them and regulatory GTPases such as the RabD class. Interactions will be achieved by fluorescence resonance energy transfer, immunoprecipitation and GST pull downs, plus the yeast two hybrid system. A yeast two hybrid screen will also be used to identify binding partners for Atp115 which is the best candidate for an ER-Golgi tether. The function of the matrix proteins will be assessed by overexpression of mutant versions with specific domain deletions, RNAi inhibition and analysis of insertional mutants. Their roles in tethering to the ER will be tested in vivo by an optical trapping approach after expression of non-functional mutants of the putative tethering proteins, whilst their role in Golgi biogenesis will be assessed by confocal microscopy of Golgi reformation after Brefeldin A or Sar1p mutant induced deconstruction of the Golgi. We will also continue to collaborate with Dr P. Dupree on identification of novel Golgi proteins by the LOPIT isotope tagging technique.