This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The endoplasmic reticulum (ER) displays extreme plasticity in a broad range of eukaryotes: the structure of the ER can vary widely in response to natural stimuli or experimental manipulations. Despite the ubiquitousness of this cellular response, little is known about the structural features or molecular underpinnings of ER plasticity. High expression levels of certain ER resident proteins cause profound proliferation of the ER membrane. The best characterized of these proteins is HMG-CoA Reductase (HMGR), which we are utilizing to rigorously examine ER plasticity in Saccharomyces cerevisiae. HMGR levels naturally fluctuate in response to the mevalonate pathway, can be increased or decreased using drugs or regulated through molecular biological mechanisms, making this an ideal system to investigate organellar dynamics. A great deal of information fundamental to understanding this biological process is attainable only by advanced light and electron microscopy techniques. For example, we can utilize electron tomography to determine if the membrane structures caused by elevated levels of HGMR are a proliferation or a structural reorganization of the ER. We can also analyze the dynamics of the system by inducing proliferations or stimulating the degradation of proliferations. Budding sites of membrane proliferations as well as structural intermediates in forming or disassembling the membrane arrays may be identified in this manner. The dynamic behavior of HMGR is completely unknown. We are able employ FRET to study the dynamics of a functional HMGR-GFP and its ability to move in and out of proliferations. Coupling the powerful genetics and biochemistry of this yeast system with advanced microscopy techniques will provide a strong platform for an enhanced understanding ER plasticity and dynamics.
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