The mechanisms of the regulation of PA biosynthesis, of the rate-limiting GPAT step, and of lipid metabolic pathway partitioning are not known. It has been generally accepted that glycerolipid biosynthetic pathways diverge at the level of PA metabolism to DAG or CDP-DAG. Our work on yeast Gat1p and Gat2p indicated that a significant partitioning of membrane biosynthetic pathways occurs at the initial GPAT-catalyzed step of lipid synthesis (32
). Likely, localization of Gat1p and Gat2p contributes to their metabolic partitioning. Thus, the first aim of this work was to carefully define the subcellular localization of these enzymes. Furthermore, since gene dosage is a powerful approach to studying Gat1p and Gat2p function, we extended our localization studies to cells depleted of one GPAT, or cells simultaneously lacking endogenous Gat1p and Gat2p, but maintained it by using the GAL1
galactose-inducible promoter to drive GAT1
expression. The data obtained from cells expressing endogenous levels of Gat1p or Gat2p presented in this study indicate that although Gat1p and Gat2p are both localized to the perinuclear ER as well as the cortical ER in exponentially growing cells, inspection of their distribution in sucrose density gradients strongly suggests that these proteins are enriched in distinct subdomains of the ER. Further evidence in support of this conclusion is that immunoaffinity precipitation of endogenous (10
) as well as overexpressed Gat1p or Gat2p (our unpublished results), followed by mass spectroscopy analysis, failed to detect the other GPAT in the precipitate, while other specific ER resident proteins were identified. Most importantly, our study unveiled a coordinated regulation between these two acylation systems, with two lines of evidence supporting this finding. First, in vivo imaging combined with subcellular fractionation analysis using sucrose density gradients of cells expressing Gat2p fused to GFP revealed changes in its ER localization and gradient distribution when Gat1p was missing. Second, Gat1p and Gat2p were basally phosphorylated in wild-type cells, but their phosphorylation status changed according to their own protein level and that of the other GPAT. In general, we noticed Gat1p and Gat2p are hyperphosphorylated when the proteins are in excess (overexpression), while they are less or not phosphorylated when there is a GPAT deficit. To our knowledge, no such regulation has been described previously for these enzymes in yeast or other organisms.
Recently, the mammalian mitochondrial GPAT (mtGPAT1) has been shown to be regulated posttranslationally via phosphorylation of residues Ser 632 and Ser 639 in its C-terminal end (3
), but the physiological relevance of these phosphorylation events remains unknown. Results from tandem mass spectrometry phosphoresidue analysis of yeast GPATs also indicate that Gat1p and Gat2p are phosphorylated in their C-terminal ends (our unpublished results). Our results do not clarify, however, whether phosphorylation regulates GPAT localization. No obvious differences in distribution of Gat1p- or Gat2p-phosphorylated species were detected in the sucrose density gradients analyzed for this study. This possibility will need to be addressed using monospecific Gat1p and Gat2p antibodies raised against phosphoresidues and immunomicroscopic analysis of their localization. Alternatively, phosphorylation may affect activity and/or half-life of the proteins. Interestingly, hyperphosphorylation of the yeast GPATs due to high levels of expression seems to be independent of their activity, since Gat1p and Gat2p mutants predicted to be catalytically dead (14
) showed the same phosphorylation status as wild-type proteins (data not shown). These results suggest that the signaling events leading to phosphorylation of these enzymes are not directly mediated by signaling lipids generated downstream of the reaction they catalyze.
represent a duplicate gene pair that was originated from a whole genome duplication event in the S. cerevisiae
lineage 100 million years ago (6
). It has been proposed that in order to persist in time, the two duplicate copies must lose full redundancy either by having at least one of the duplicate copies gain a new function or by partitioning the ancestral function (27
). Redundancy of GPATs is even higher in multicellular organisms. Do the various isoforms have distinct functions? Mammalian GPAT1, -2, and -3 have been shown to function mainly in triacylglycerol synthesis (12
), and just recently, GPAT4 has been found to be different, as it is a major contributor of phospholipid biosynthesis (7
). With the goal of learning more about the scope of cellular processes regulated by each GPAT, we explored the effect that overexpression of Gat1p or Gat2p had on the extreme case of cells lacking endogenous GPATs. Gat1p and Gat2p are predicted to be transmembrane enzymes, but unlike overexpression of Hmg1p, they do not form arrays of membranes around the nucleus (known as karmellae) in order to accommodate the excess of protein, when overexpressed (17
). Instead, a unique ER arrangement for dko-GAT1
cells was observed at the ultrastructural level (Fig. ). These arrays frequently enclosed or grouped mitochondria, explaining the DAPI stain trapped in the membranous mesh detected by immunofluorescence microscopy of Gat1p and Gat2p. It will be interesting to study how Gat1p and Gat2p affect mitochondrial lipid synthesis, composition, and function. In fact, growth of these dko strains on galactose, a semifermentative carbon source, is abnormally slow compared to that of its isogenic wild type.
Interestingly, gat1Δ gat2Δ
double-knockout cells overexpressing Gat1p or Gat2p (grown on galactose) show remarkable morphological differences. Elongated cells were the hallmark of dko-GAT1
strains, while large and round cells were the predominant morphology of dko-GAT2
cells. Analysis of gat1Δ
single-deletion strains grown on galactose showed morphological characteristics of dko-GAT2
strains, respectively. Importantly, we did not detect defects in nuclear division. Thus, we propose the imbalance in Gat1p and Gat2p levels in these cells affects the morphogenesis checkpoint, producing a delay in the apical isotropic switch (elongated cells) or rushing through it (larger and rounder cells). It is worth noting that these phenotypes were clearly observed in strains derived from the W303 background but not in BY4741-derived deletion strains. Overexpression of Gat1p or Gat2p in wild-type BY4741 cells showed the same morphological trend when grown on galactose, but the phenotype was not as obvious. This strain-dependent difference in cell morphology could be due to the known polymorphism at the SSD1
locus between these two strains (30
). In addition, W303 has a bud4
mutation, and both Bud4p and Ssd1p have been shown to have profound effects on the yeast morphogenesis network (30
). We have not yet explored possible genetic interactions between GAT1
with these genes.
Despite the differences between genetic backgrounds, our results show unique phenotypes when an important imbalance in Gat1p and Gat2p was induced. A major structural difference between the two dko strains was found with the cortical ER morphology. In the case of dko-GAT2
cells, the cortical ER was very irregular and also affected the PM organization, presenting frequent invaginations with multilayered membranous structures. Cortical ER inheritance has been shown to be required for normal septin organization and polarized growth (21
). It is possible that alterations in the cortical ER observed in dko-GAT1
cells differentially affect ER compartmentalization and, consequently, cell polarity. This effect on the PM and cortical ER could also explain the change in the equilibrium distribution observed for Pma1p when Gat2p was the only GPAT present in the cell. A synthetic sick interaction between a deletion of GAT2
and a PMA1
allele that yields reduced levels of this essential protein (DAmP strain) has been described previously (28
), further supporting a role of the acyltransferase in Pma1p localization and/or function.
Lastly, the complete lack of GPATs causes cell death, with multibudded cells containing a nucleus. In fact, we have the impression that these cells have recycled their organelles' membranes through an autophagy-like process in order to feed and maintain their nuclear envelopes, as judged by the electron microscopy images of these cells showing multivesiculated arrangements decorating the nuclear envelope surroundings. This terminal phenotype could be indicative of problems in postcytokinesis processes that lead to cell separation. We do not know at this point if it is related to a cell cycle defect or a deficiency in the delivery of degradative enzymes to the cell wall.
Together, these findings have several interesting implications. They uncover a mechanism of cellular compensation in response to abnormalities in the first step of acylation, forming LysoPA that impacts not only lipid metabolism but other cellular processes that are coupled to it, like polarized cell growth and cell separation. Future efforts will focus on understanding the contribution of each GPAT to these processes. They include identification of phosphoresidues in Gat1p and Gat2p as well as the signaling pathway(s) involved in their phosphorylation. In addition, the molecular mechanisms underlying distinct contributions of Gat1p and Gat2p to the formation of ER subdomains and their protein composition are the subject of future investigations.