Vladimir DenicDepartment of Molecular and Cellular Biology
Northwest Laboratories Bldg. Room 445.3
52 Oxford St, Cambridge, MA 02138
Research Interests and Goals:
Molecular Principles Enabling Structural Diversification of Very Long-Chain Fatty Acids.Very long-chain fatty acids (VLCFAs) are structurally diverse biological molecules with unusually long hydrocarbon chains, ranging from 20 to 36 carbons (C20-36) or more. These lipids perform essential roles in a wide range of biological processes that cannot be performed by the more common shorter fatty acids (i.e., C16 and C18). For example, the chain length of certain VLCFAs allows them to simultaneously reside in both leaflets of the lipid bilayer, thereby stabilizing highly curved cellular membranes, such as those surrounding the nuclear pore complexes. Notably, the variation in VLCFA chain length across different species and different tissues has enabled the numerous functional specializations of these lipids. In S. cerevisiae, for example, C26 is specifically required for a variety of membrane-based processes, including the formation of GPI lipid anchors and trafficking of proteins in the secretory pathway. Similarly, in mammals VLCFAs with lengths >C30 are critical for the permeability barrier of the skin while others act as signaling molecules or as the dominant lipid constituents of certain tissues (e.g., photoreceptor cells and myelin).
I have recently developed a defined in vitro system for studying VLCFA synthesis. This allowed me to demonstrate that the chain-length diversity of cellular VLCFAs originates with a series of related membrane protein complexes, each comprising a common catalytic core and a specific elongase protein (Elop). These complexes catalyze processive rounds of two-carbon that culminate with the release of defined length products. Additionally, I was able to show that Elops are a novel class of membrane-embedded condensing enzymes that have a fixed catalytic site and a physically distinct region that has been subject to evolutionary diversification. Lastly, I uncovered a mechanism by which yeast Elop homologs specify the length of VLCFA molecules using an intramembrane alpha-helical measuring strategy and then exploited this understanding to rationally design Elops with dramatically altered substrate specificities.
In future studies, I plan to characterize how Elops are able to catalyze the chemically well-understood Claisen condensation reaction while using novel sequence requirements suggestive of metal ion-assisted catalysis. Additionally, I wish to exploit the ability of my in vitro system to work efficiently with Elop homologs from distant organisms as a means of uncovering novel Elop measuring strategies underlying VLCFA diversity. A great starting point in this regard are metazoan Elops required for the elongation of hydrocarbon chains with other topologies, such those containing multiple double bonds (e.g., the omega-3 fatty acids) and mono-methyl branches. Lastly, I wish to understand the structural basis of the evolutionary malleability of the Elop fold that has allowed it to form intramembrane pockets for guiding the iterative polymerization of substrates with widely different lengths, flexibilities, and geometries.
The Mechanism of Tail-Anchored Protein Insertion into the Endoplasmic Reticulum Membrane.Tail-anchored (TA) proteins are defined by the presence of a single transmembrane domain (TMD) located near their C-termini that tethers them to different internal membranes while allowing them to present their functional N-terminal domains to the cytosol. TA proteins are found throughout the secretory pathway, in the nuclear envelope, peroxisomes, and mitochondria. Within the secretory pathway, TA proteins play diverse roles, such as enabling vesicular traffic (e.g., many of the SNAREs, which mediate fusion of secretory vesicles, are TA proteins), aiding in protein translocation, and promoting folding or degradation of membrane proteins. The biogenesis of secretory pathway TA proteins involves first insertion into the endoplasmic reticulum (ER) membrane followed by sorting to their final destinations (including the nuclear envelope and peroxisomes). In contrast, mitochondrial TA proteins are inserted directly into the outer mitochondrial membrane where they facilitate mitochondrial fission and act in apoptosis.
Using a genetically-tractable yeast cell-free system, I have recently demonstrated that Get1/2/3 are the molecular machinery directly responsible for the insertion of secretory pathway TA proteins into the ER membrane. Specifically, I have shown that the soluble ATPase Get3 recognizes TMDs of ER-destined TA proteins but not those of mitochondrial TA proteins. This recognition event represents a key decision step, as loss of Get3 can lead to misinsertion of secretory pathway TA proteins into mitochondria. Lastly, I have found that Get3-TA complexes become recruited for ER membrane insertion by a transmembrane complex consisting of Get1 and Get2.
In future studies, I plan to take advantage of my in vitro system to address several key mechanistic issues. What determines whether a TA protein is inserted into the ER or the mitochondrial membrane? Operationally, the targeting information appears to be largely encoded in the TMDs and flanking regions, but the precise biophysical determinants distinguishing ER from mitochondrial TMDs and how they get decoded by Get3, are not well understood issues. Given that some secretory pathway TA proteins are inherently symmetrical and become readily misinserted into mitochondria in the absence of Get3, what mechanisms ensure that under normal conditions they get efficiently “channeled” to the ER following their release from the ribosome? How is ATP hydrolysis used by the GET system to overcome the energetic barriers to spontaneous insertion of transmembrane proteins into lipid bilayers?
Wang, F., Whynot, A., Tung, M., and Denic, V. (2011) The Mechanism of Tail-Anchored Membrane Protein Insertion into the ER Membrane. Mol Cell (in press).
Wang F., Brown, E.C., Mak, G., Zhuang, J., and Denic, V. (2010) A Chaperone Cascade Sorts Proteins for Posttranslational Membrane Insertion into the Endoplasmic Reticulum. Mol Cell, 8 (40): 159-171.
Stefer, S., Reitz, S., Wang, F., Wild, K., Pang, Y.Y., Schwarz, D., Bomke, J., Hein, C., Löhr, F., Bernhard, F., Denic, V., Dötsch, V., and Sinning I. (2011) Structural Basis for Tail-Anchored Membrane Protein Biogenesis by the Get3-Receptor Complex. Science, 333(6043): 758-62. Epub 2011 June 30.
Schuldiner, M., Metz, J., Schmid, V., Denic, V. et al. (2008) The Get Complex Mediates Insertion of Tail-Anchored Proteins into the ER. Cell; 134: 634-45.
Denic, V., Weissman J.S. (2007) A Molecular Caliper Mechanism for Determining Very Long-Chain Fatty Acid Length. Cell; 130: 663-677.
Denic, V., Quan, E.M., and Weissman, J.S. (2006) A Luminal Surveillance Complex that Selects Misfolded Glycoproteins for Endoplasmic Reticulum-Associated Degradation. Cell; 126: 349-359.
Schuldiner, M., Collins, S.R., Thompson, N. J., Denic, V. et al. (2005) Exploration of the Function and Organization of the Yeast Early Secretory Pathway through an Epistatic Miniarray Profile. Cell; 123: 507-519.
Bhamidipati, A., Denic, V., Quan, E.M., and Weissman, J.S. (2005) Exploration of the Topological Requirements of ERAD Identifies Yos9p as a Lectin Sensor of Misfolded Glycoproteins in the ER lumen. Molecular Cell; 19: 741-751.
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