The Spatial Organization of Lipid Synthesis in the Yeast Saccharomyces cerevisiae Derived from Large Scale Green Fluorescent Protein Tagging and High Resolution Microscopy

Abstract

The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins (ϳ9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi. Molecular & Cellular Proteomics 4:662-672, 2005.Biological membranes are characterized by a highly complex mixture of lipids that are key determinants of the biophysical parameters of membranes, such as fluidity, permeability, and signaling functions. Thus, lipids affect structure and function of peripheral and integral membrane proteins, and thus play a pivotal role in organelle (membrane) function. In the yeast Saccharomyces cerevisiae, the lipid composition appears rather simple, consisting mainly of glycerophospholipids, sphingolipids, and ergosterol (1); however, recent advances in analytical techniques such as nanoelectrospray ionization tandem mass spectrometry (2) have unveiled a great degree of heterogeneity within the molecular species of phospholipids. Furthermore subcellular membranes differ not only in their lipid composition in terms of lipid classes, they are also characterized by a distinct distribution of molecular species of phospholipids (3). Because only a few membranes harbor enzymes involved in lipid synthesis, this heterogeneity may be established by localized synthesis, localized degradation, and selective trafficking of membrane lipids. Interestingly apparently "linear" pathways for the synthesis of major membrane lipids involve multiple organelles: the synthesis of phosphatidylserine by a synthase (Cho1p) occurs in the endoplasmic reticulum (ER) 1 and is followed by decarboxylation to phosphatidylethanolamine in the mitochondrial inner membrane (by Psd1p) or Golgi/vacuoles (by Psd2p). Subsequently phosphatidyletha...

Loss of Ypk1, the Yeast Homolog to the Human Serum- and Glucocorticoid-induced Protein Kinase, Accelerates Phospholipase B1-mediated Phosphatidylcholine Deacylation

“…3C). These results, which are consistent with fluorescence microscopy of C-terminally tagged Plb1 under the control of the TEF promoter (62), lead us to conclude that the protein is not plasma membrane-associated and that if it is GPIanchored, it is readily released from the plasma membranes under the conditions used here. We suspect that the cell surface-associated Plb1 activity that releases GroPCho into the medium is due to its periplasmic localization.…”

“…However, localization data were absent in large scale analyses using fluorescent tags fused to the C termini of yeast ORFs expressed under control of their endogenous promoters (56 -60) and in a study utilizing C-terminal fusions to an epitope tag with the target genes expressed under control of the GAL promoter (61). In addition, a localization screen of proteins involved in lipid metabolism, in which Plb1 was C-terminally tagged and expressed under the control of the TEF promoter, demonstrated ER and vesicle localization, but plasma membrane localization was not apparent; the medium/secreted fraction was not analyzed in this study (62). Complicating the question of Plb1 localization is that it has a predicted GPI anchor, which should lead to plasma membrane residence (63,64).…”

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

“…In parallel, we added to yL405 a version of the second FAR fused at the Cterminus to a green fluorescent protein (GFP) to examine enzyme solubility and localization. Confocal microscopy showed GFP localization in a pattern typical of the endoplasmic reticulum (ER) common in lipid metabolic enzymes (Natter et al, 2005) (Fig. 2B).…”

“…The bars represent the mean, and the error bars one standard deviation, for three to four biological replicates. (B) Confocal microscopy images of yL405 containing MmFAR1-GFP shows the fusion enzyme to be ER-localized, as are many native enzymes that use the same substrate fatty acyl-CoA (Natter et al, 2005). Fig.…”

Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry

“…Despite the extensive characterization of proteins, their association into complexes and activities (4)(5)(6), it is still difficult to assess how perturbations within the lipid metabolic network affect the full lipidome of cells. This work shows that lipidome-wide quantification of individual molecular lipid species (molecules with defined chemical structure) by absolute quantification (expressed in mol or mol%) provides a new approach to relate lipidomics and functional genomics studies.…”