Functional and Topological Analysis of Acyl-CoA:Diacylglycerol Acyltransferase 2 From Saccharomyces cerevisiae

• Author / Creator

  Liu, Qin

• Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20, DGAT or DAGAT) is a membrane protein found mainly in the endoplasmic reticulum (ER). It catalyzes the final step in the biosynthesis of triacylglyerol (TAG or TG), which is the principal repository of fatty acids for energy utilization and membrane formation. Several lines of evidence have indicated that DGAT has a substantial effect on carbon flux into TAG. DGAT has at least two discrete family members (DGAT1 and DGAT2) with different physiological roles. High-resolution structures of both DGATs, however, are absent due to difficulties in purification. In order to gain insight into structural and functional relationships of DGATs, a functional DGAT2 protein from the yeast Saccharomyces cerevisiae (ScDGAT2, also known as Dgalp) was selected. The structural and functional role of cysteine residues in ScDGAT2 was studied using site-directed mutagenesis (SDM) in combination with chemical modification. Although ScDGAT2 is susceptible to thiol-modifying reagents, none of the cysteines are essential for the catalytic activity or involved in structure support though disulfide linkages. Inhibition of DGAT activity by thiol-specific modification was localized to cysteine314, which is in the proximity of a highly conserved motif in DGAT2s. Thus, cysteine314 may reside in a crucial position near a possible active site or related to proper protein folding. The functional importance and topological orientation of signature motifs in ScDGAT2 were also studied using the same methods. Both the N- and C-termini of ScDGAT2 are oriented toward the cytosol. A highly conserved motif, 129YFP131, and a hydrophilic segment exclusive to ScDGAT2, reside in the ER and play essential roles in enzyme catalysis. In addition, the strongly conserved H195, which may be part of the active site of DGAT2, is likely embedded in the membrane. Although ScDGAT2 has a topology similar to that of murine DGAT2, there are striking differences which suggest that the topological organization of DGAT2 is not ubiquitously conserved.

• Subjects / Keywords

  • Structure/Function
  • Topology
  • Mutagenesis
  • DGAT2
  • TAG
  • Yeast

• Graduation date

  Spring 2011

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3734K

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.