Title page for ETD etd-3331171059721601


Type of Document Dissertation
Author Denbow, Cynthia Jones
Author's Email Address cdenbow@vt.edu
URN etd-3331171059721601
Title Membrane Domain of Plant 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase: Targeting, Topology, and Function
Degree PhD
Department Plant Pathology, Physiology, and Weed Science
Advisory Committee
Advisor Name Title
Grabau, Elizabeth A.
Hatzios, Kriton K.
Radin, David N.
Tolin, Sue A.
Cramer, Carole L. Committee Chair
Keywords
  • protein degradation
  • membrane topology
  • protein targeting
  • tomato HMGR
  • epitope-tag
  • transgenic cells
Date of Defense 1997-05-06
Availability unrestricted
Abstract
The rate limiting step in isoprenoid biosynthesis is catalyzed by 3-hydroxy-3-methylglutaryl CoA reductase (HMGR, EC 1.1.1.34). In plants, HMGR is encoded by small gene families whose members are differentially expressed. In tomato, hmg2 was previously isolated and sequenced. We report the isolation and sequence analysis of a clone (pCD4) encompassing exon I of tomato hmg1 which encodes the putative membrane domain. Sequence comparisons of plant HMGR proteins reveal two hydrophobic stretches within the amino terminus which are highly conserved among species. Using in vitro transcription and translation systems, the membrane domain structure of two tomato HMGR isoforms, HMG1 and HMG2, were analyzed. Results from these experiments reveal that tomato HMGRs are targeted to microsomal membranes in a cotranslational fashion that does not involve cleavage of an N-terminal targeting peptide. Membrane topography of HMGR was revealed by protease protection studies, indicating that both tomato HMGRs span the membrane two times such that both the C- and N-termini are located within the cytosol. HMG2 but not HMG1 was glycosylated in the in vitro system. Deletion of the hmg1 5' untranslated regions and sequences encoding the first six highly charged amino acids resulted in inefficient translation in vitro. However, targeting to microsomes was unchanged. HMG1 membrane domain was tagged with a FLAG epitope to facilitate in vivo studies. Agrobacterium-mediated transformation was used to introduce the tagged hmg1 gene into two Nicotiana tabacum cell lines, BY-2 and KY-14. The slow growth kinetics of KY-14 prevented effective recovery of transformed lines, however, Northern analyses of BY-2 showed that the hmg1 transgene was expressed. Comparisons of BY-2 and KY-14 revealed differences in defense responses to elicitor treatment. BY-2 cells showed minimal defense capabilities, whereas KY-14 cells were rapidly induced as indicated by increased HMGR enzyme activity and browning of the cells. HMGR enzyme activity was decreased in both KY-14 and BY-2 cells following sterol treatment, but the reduction was more pronounced in KY-14 cells. Thus transgenic BY-2 cells may be useful in future in vivo immunolocalization studies, but analyses of HMGR transcriptional regulation and regulated degradation will require use of the more responsive KY-14 cells..

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