Cells regulate the biophysical properties of their membranes by coordinated synthesis of different classes of lipids. Here, we identified a highly dynamic feedback mechanism by which the budding yeast Saccharomyces cerevisiae can regulate phospholipid biosynthesis. Phosphatidic acid on the endoplasmic reticulum directly bound to the soluble transcriptional repressor Opi1p to maintain it as inactive outside the nucleus. After the addition of the lipid precursor inositol, this phosphatidic acid was rapidly consumed, releasing Opi1p from the endoplasmic reticulum and allowing its nuclear translocation and repression of target genes. Thus, phosphatidic acid appears to be both an essential ubiquitous metabolic intermediate and a signaling lipid.

Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl-tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents.

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to generate sphingosine 1-phosphate (S1P). Because both substrate and product of the enzyme are potentially important signaling molecules, the regulation of SK1 is of considerable interest. We report that SK1, which is ordinarily a cytosolic enzyme, translocates in vivo and in vitro to membrane compartments enriched in phosphatidic acid (PA), the lipid product of phospholipase D. This translocation depends on direct interaction of SK1 with PA, because recombinant purified enzyme shows strong affinity for pure PA coupled to Affi-Gel. The SK1-PA interaction maps to the C terminus of SK1 and is independent of catalytic activity or of the diacylglycerol kinaselike domain of the enzyme. Thus SK1 constitutes a novel, physiologically relevant PA effector.The potential function of PA 1 in signal transduction is not well established, and there is even debate about whether PA itself (or a downstream metabolite) is a signaling lipid (1). Whereas many cellular control pathways exhibit a stimulusdependent elevation of PLD activity (arguing for a direct role of the lipid product of PLD-PA in these pathways (2)), historically, the search for classic protein effectors of PA has failed to yield many clear-cut examples where a specific response to elevated PA levels could be demonstrated directly.Recent data from a number of laboratories are beginning to define some potential PA targets. These include kinases (e.g. Raf-1 (3, 4) and mammalian target of rapamycin (5)), phosphatases (e.g. protein-tyrosine phosphatase SHP-1 (6) and protein phosphatase-1 (7)), enzymes involved in lipid turnover (e.g. phosphatidylinositol-4-phosphate 5-kinase (8, 9) and phospholipase C (10)), as well as other enzymes (e.g. phosphodiesterase PDE4D3 (11, 12)). There are additional examples of proteins regulated by PA in combination with other lipids (e.g. protein kinase C⑀ (13) and p47phox (14)). With the possible exception of Raf-1 and PKC⑀, regulation of the other proteins by PA has not been shown to result directly in membrane translocation in vivo. Raf-1 accumulates in endosomal membranes as a consequence of PA formation (4), whereas PKC⑀ translocates to the plasma membrane in response to coordinated production of PA and diacylglycerol (13).By phosphorylating sphingosine to generate S1P, SK1 regulates the levels of two important bioactive lipids and affects a number of cellular functions, including for example calcium mobilization, growth control, and cytoskeletal rearrangements among others (for a recent review see Ref. 15). We became interested in the possibility that SK1 may constitute a novel PA effector based on three types of evidence. SK1 activity is stimulated by acidic phospholipids, including PA (16), it is found up-regulated in immune cells following PLD activation (reviewed in Ref. 17), and the SK1 protein is activated by PKC (a PLD activator) and translocates to membranes following stimulation with phorbol esters (18).In this work we show that SK1 is a PA effector and responds to PA f...

Posttranslational modification of proteins with farnesyl and geranylgeranyl isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three protein prenyltransferases: farnesyl transferase (FTase), geranylgeranyl transferase type-I (GGTase-I), and Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these enzymes have emerged as promising therapeutic compounds for treatment of cancer, viral and parasite originated diseases, as well as osteoporosis. However, no generic nonradioactive protein prenyltransferase assay has been reported to date, complicating identification of enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases.