Structure and Inheritance of the endoplasmic reticulum

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In all eukarotic cells, the endoplasmic reticulum (ER) forms a highly fenestrated tubular network that spreads throughout the volume of the cell, often lying just below the plasma membrane. This distribution may be critical for calcium signalling and other important cellular functions. We have taken a genetic approach towards understanding the mechanism of ER dynamics, structure and inheritance in yeast. Our work to date shows that ER tubules form from the nucler envelope at the start of the cell cycle, they are delivered into the bud along actin cables by the type V myosin, Myo4p, they are anchored at the bud tip by the exocyst complex and then spread along the cell cortex to form the cortical ER. We propose that Rtn1 p, a member of the conserved reticulon family of proteins, functions as the receptor for the exocyst subunit Sec6p on the ER membrane and that Rtn1 p also serves to link the ER to the cell cortex. Four specific aims are planned: 1) We will analyze the interaction of Rtnlp with the exocyst, defining the topology of Rtnlp within the ER membrane and the specific interactions that link the exocyst to Rtnlp. 2) We will determine if Rtnlp binds to the Tcb3 protein along the cortex and if this interaction underlies the unique localization of Rtnlp as well as the tight association of the ER with the cell cortex. 3) We will determine if Rtnlp plays a role in forming the fenestrated reticulum characteristic of the ER in all eukaryotes and if it interacts with other components of the ER membrane in this capacity. 4) We will express or microinject dominant negative alleles of mammalian reticulon proteins as well as make use of RNAi methodology to inhibit the function of the four mammalian RTN proteins. Phenotypic analysis will determine if the roles of Rtnlp and its interactions are conserved.