|Gidda, Satinder - U OF GUELPH, GUELPH, ON|
|Rothstein, Steven - U OF GUELPH, GUELPH, ON|
|Mullen, Robert - U OF GUELPH, GUELPH, ON|
Submitted to: Plant Physiology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 27, 2009
Publication Date: June 9, 2009
Citation: Gidda, S.K., Shockey, J.M., Rothstein, S.J., Dyer, J.M., Mullen, R.T. (2009). Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals: functional divergence of the dilysine ER retrieval motif in plant cells. Plant Physiology and Biochemistry, 47:867-879. Interpretive Summary: Lipids are vital components of all living cells that serve many different functions in plants including the formation of the protective, waxy layer on the surface of plant leaves and the accumulation of storage oils (vegetable oils) in developing seeds. As such, lipids protect plants from environmental stress and also serve as important components of human nutrition. Lipids are produced by complex metabolic pathways in plant cells, and developing a better understanding of the relevant enzyme activities is important for establishing a knowledge base that can be used to improve the characteristics and/or nutritional value of plants. Here we provide an in-depth analysis of two enzymes that each perform the first step of lipid synthesis in plant cells. We describe in detail exactly where in plant cells these enzymes reside, how they get to that location, and how they are maintained at that position. The information gained from these studies provided insight not only to the enzymes of lipid production, but also to other enzymes that are found in this same location within plant cells. The results of this study will have a broad impact on those scientists that are interested in understanding how certain enzymes (and their associated enzyme activities) are organized in plant cells to carry out specific metabolic functions.
Technical Abstract: Glycerol-3-phosphate acyltransferase (GPAT; EC 18.104.22.168) catalyzes the committed step in the production of glycerolipids, which are major components of cellular membranes, seed storage oils, and epicuticular wax coatings. While the biochemical activities of GPATs have been characterized in detail, the cellular features of these enzymes are only beginning to emerge. Here we characterized the phylogenetic relationships and cellular properties of two GPAT enzymes from the relatively large Arabidopsis thaliana GPAT family, including GPAT8, which is involved in cutin biosynthesis, and GPAT9, which is a new putative GPAT that has extensive homology with a GPAT from mammalian cells involved in storage oil formation and, thus, may have a similar role in plants. Immunofluorescence microscopy of transiently-expressed myc-epitope-tagged GPAT8 and GPAT9 revealed that both proteins were localized to the endoplasmic reticulum (ER), and differential permeabilization experiments indicated that their N and C termini were oriented towards the cytosol. However, these two proteins contained distinct types of ER retrieval signals, with GPAT8 possessing a divergent type of dilysine motif (-KK-COOH rather than the prototypic -KKXX-COOH or -KXKXX-COOH motif) and GPAT9 possessing a hydrophobic pentapeptide motif (-phi-X-X-K/R/D/E-phi-; where phi are large hydrophobic amino acid residues). Notably, the divergent dilysine motif in GPAT8 only functioned effectively when additional upstream residues were included to provide the proper protein context. Extensive mutational analyses of the divergent dilysine motif, based upon sequences present in the C termini of other GPAT8s from various plant species, also further expanded the functional definition of this molecular targeting signal, thereby providing insight to the targeting signals in other GPAT family members as well as other ER-resident membrane proteins within plant cells.