Cragun, David2013-07-232016-06-012011-01-31bibid: 8213802https://hdl.handle.net/1813/33508A defining feature of eukaryotic life is the presence of membrane-bound organelles. While the energetically most stable shape for a membrane is likely a sphere, many organelles possess much more complex shapes within cells which contributes to their proper function. There has been an explosion in the field of membrane bending proteins in recent years giving new insights into how a cell forms and maintains organelle structures. Two ER resident proteins have recently been implicated in the formation of the tubular network of the ER in both higher and lower eukaryotes. Both Rtn1p and Yop1p contain two long hydrophobic domains, ~40 amino acids in length, which are thought to act as wedges within the outer leaflet of the ER membrane and drive membrane deformation. Using a mutagenic approach I have determined that both of the hydrophobic domains of Yop1p are critical for its ability to generate the tubules of the peripheral ER. The overexpression of Yop1p produces long, unbranched tubules in cells. I have developed an initial strategy for the enrichment of these Yop1p formed tubules for further study outside the context of the cell. Biochemical analysis of these tubular structures has revealed they are composed of protein and lipid components. Electron microscopy of enriched tubules indicates they have a small diameter, ~15 nm, and are often bundled together into rope-like structures. This work underscores the importance of Yop1p in the generation of the tubular network of the peripheral ER and provides evidence deepening our understanding of the molecular mechanism of Yop1p action on membranes that generates membrane tubules.en-USmembranecurvatureyop1dissertation or thesis