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Title: 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

Author
item GIDDA, SATINDER - University Of Guelph
item Shockey, Jay
item ROTHSTEIN, STEVEN - University Of Guelph
item Dyer, John
item MULLEN, ROBERT - University Of Guelph

Submitted to: Plant Physiology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/27/2009
Publication Date: 6/9/2009
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(10):867-879.

Interpretive Summary: Our laboratories, in addition to many others in government, academia, and private industry seek to create new types of oilseed crops that produce oils with value-added traits. These new oils could replace petroleum-based compounds in many everyday products such as nylons, plastics, inks, and dyes. One way to accomplish this goal is to engineer common oilseed plants, such as cotton, soybean, canola to contain foreign genes from other plants that produce the value-added oils. However, many different enzymes can influence the level and types of oils made in seeds, while other closely related enzymes may play no role at all in oil production. Development of methods to distinguish the useful genes from their non-useful counterparts is therefore essential. One of the properties of a useful form of an enzyme is one that is directed to the specific parts of the cells in oilseeds where oil synthesis occurs. Only small portions of an otherwise large enzyme are typically needed to direct the enzyme to its proper destination in a cell. However, determining the location and limits of these small portions can sometimes be very difficult. The present manuscript describes the characterization of the necessary portions of two glycerol-3-phosphate acyltransferases (GPAT) enzymes, called GPAT8 and GPAT9, found in developing seeds of the Arabidopsis thaliana, a model oilseed crop whose genetics, biochemistry and cellular structure closely resembles that of many other valuable industrial oil-producing plant species.

Technical Abstract: Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) 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 (-F-X-X-K/R/D/E-F-); where F 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, 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.