Phosphoinositides are well-known components of cellular signal transduction pathways and, more recently, have been shown to play important roles in organelle identity and targeting determinants for various cytosolic proteins. Conversion of PtdIns into its various phosphorylated derivatives, such as PtdIns4P and PtdIns(4,5)P2, is accomplished by a series of distinct lipid kinase and lipid phosphatase activities that are localized to specific subcellular membranes. As a result, production of distinct PtdIns forms is thought to be largely dependent on the access of these enzymes to their PtdIns or PtdInsP substrates. Interestingly, an investigation of two different PIS (PtdIns synthase) isoforms by Lofke et al. in this issue of the Biochemical Journal now indicates that the ability of PtdIns to be converted into downstream PtdInsPs may depend upon the PIS isoform from which it was synthesized.

A typical eukaryotic cell contains a myriad of distinct membrane types, each providing unique properties essential for proper functioning of the cell. The functional distinctions between these membranes are, at least in part, due to the unique composition of lipids found within that particular membrane. In addition to affecting physical properties of the membrane, several classes of lipids are known to serve as intracellular second messengers and play integral roles in transduction of signals from a host of different cellular processes. Most prominent among these signalling lipids is PtdIns and its complement of short-lived phosphorylated derivatives, the PtdInsPs [1].

PtdIns and its associated PtdInsPs are relatively minor forms of the phosphoacylglycerol family of lipids, and usually comprise less than 10% of the total lipid content in any biological membrane. As with other phosphoacylglycerols, PtdIns consists of three parts: a three-carbon glycerol backbone, two long-chain fatty acids attached to the C-1 and C-2-positions via ester bonds and an inositol sugar attached to the C-3-position of the glycerol molecule via a phosphodiester bond. The PtdIns lipid itself is synthesized from addition of a myo-inositol sugar head group to activated CDP-DAG (CDP-diacylglycerol) with the resultant formation of PtdIns and CMP [2]. This synthesis is carried out by specific PISs (PtdIns synthases), which have been characterized in animals, fungi and plants and are thought to act at the interface between the ER (endoplasmic reticulum) and Golgi membrane compartments [3]. Typically, PISs are ~26 kDa enzymes that contain conserved CDP-alcohol PtdIns transferase domains attached to a series of four transmembrane domains. In subsequent steps, the various phosphorylated PtdInsPs are then generated by the combined activities of a series of phosphoinositide kinases and phospho-inositide phosphatases that act either separately or in concert on the D-3, D-4 and D-5 positions of the inositol headgroup. So far, seven distinct PtdInsP forms have been identified in eukaryotic membranes and named on the basis of their site(s) of phosphorylation: PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns4,5P2 and PtdIns(3,4,5)P3 [1].

Classically, PtdInsP involvement in signalling pathways focused on the cleavage of PtdIns(4,5)P2 into soluble Ins(3,4,5)P3 and membrane-associated DAG second messengers. However, previous studies have revealed that the localized accumulation of various PtdInsP isoforms within the cell probably plays as important a role in cellular signalling as the production of Ins(3,4,5)P3 and DAG second messengers. Specific accumulation of PtdInsP isoforms has been shown to help specify organelle identity and to serve as targets for recruitment of elements of the regulatory machineries that control dynamics of both membrane trafficking and the cytoskeleton [4]. These studies have led to the increased recognition that where these lipids accumulate within the cell is as important as when they accumulate. Accordingly, it has become clear that there are often multiple forms of the enzymes that phosphorylate or dephosphorylate the PtdIns inositol ring at any specific position, and that different enzymes can be responsible for producing specific PtdInsP isoforms in distinct subcellular pools within the cell.

A clear example of how similar phosphoinositide kinase activities are carried out by distinct enzymes with non-overlapping functions can be found in the generation of PtdIns4P and PtdIns(4,5)P2 during membrane trafficking and signalling pathways in yeast and mammalian cells. In all eukaryotic cells studied so far, generation of PtdIns4P occurs through the action of PI4Ks (phosphoinositide 4-kinases) which phosphorylate the inositol head group of PtdIns at the D-4 position, resulting in PtdIns4P. Two main types of PI4K (Type II and Type III) have been classified in yeast, mammalian and plant cells. In yeast, the single Type II PI4K, the LSB6 gene product, is non-essential, and elimination results in only minor decreases in PtdIns4P production [5]. In contrast, both yeast Type III PI4Ks are essential and provide distinct functions, as deletion of either gene is lethal and cannot be rescued with overexpression of the other form. This essential nature is reflected by distinct subcellular distributions of these two PI4Ks, with the high-molecular-mass PI4Kα orthologue, the STT4 gene product, found at the plasma membrane, whereas the yeast PI4Kβ orthologue, the PIK1 gene product, localizes to the Golgi complex and nuclear envelope membranes [6]. Interestingly, in mammalian cells, both the Type II PI4KIIα and Type III PI4KIIIβ enzymes are localized to trans-Golgi cisternae and TGN (trans-Golgi network) complexes, but again appear to provide non-overlapping functions, as suppression of either of these PI4K activities appears to affect distinct steps during membrane trafficking through these membrane compartments [7]. Similarly, generation of distinct subcellular pools of PtdIns(4,5)P2 also occurs by the subsequent conversion of PtdIns4P into PtdIns(4,5)P2 by distinct PIP5Ks (PtdIns4P 5-kinases). At the plasma membrane, PtdIns(4,5)P2 accumulation drives both clathrin-dependent and clathrin-independent endocytic events, and it is likely that different PIP5K isoforms are selectively recruited and activated to locally convert PtdIns4P into PtdIns(4,5)P2 to carry out these processes [8].

Another important factor that regulates the ability to accumulate various PtdInsPs within the cell is the availability of the initial PtdIns substrate in any subcellular compartment. Although PtdIns is initially thought to be synthesized by PIS at the ER/Golgi interface, PITPs (PtdIns transfer proteins) are capable of mobilizing PtdIns from one subcellular membrane to another, and thereby help regulate the discrete pools of PtdIns available in any particular biological membrane. The central role of PITP proteins in regulation of PtdInsP production is demonstrated by the essential nature of the yeast PITP, the SEC14 gene product, in yeast [9]. Arabidopsis contains a large multigene family of SEC14 homologues, which play important roles in polarized secretion [10]. Furthermore, of the three mammalian SEC14 homologues, two are localized to the Golgi complex and are required for regulated exocytosis and intra-Golgi transport steps [11].

In this issue of the Biochemical Journal, Löfke et al. [12] demonstrate that PIS2, which shows similarity to the previously characterized Arabidopsis PIS1, is an active enzyme capable of producing PtdIns in vitro. Both PIS1 and PIS2 localized to ER and Golgi membranes, and stable overexpression of these two PIS isoforms resulted in plants that accumulated higher levels of PtdIns, indicating that each of these isoforms is capable of generating PtdIns in vivo. Surprisingly, only plants overexpressing PIS2 displayed additional significant increases in PtdIns4P and PtdIns(4,5)P2. In contrast, the elevated PtdIns levels in plants overexpressing PIS1 were associated with elevated DAG and phosphatidylethanolamine levels, but displayed no change in PtdIns4P and PtdIns(4,5)P2. Further examination of the fatty acid complements of the pools of PtdIns generated by PIS1 and PIS2 in these transgenic plants revealed that PIS1 selectively added saturated or monounsaturated fatty acids, whereas PIS2-overexpressing plants accumulated higher levels of unsaturated fatty acids incorporated into the increased PtdIns pool.

The results presented by Löfke et al. [12] raise the intriguing possibility that PtdIns synthesized from distinct PIS isoforms may form distinct pools: one which is readily converted by PI4K and PIP5K to PtdIns4P and PtdIns(4,5)P2, and a second pool which is not. Previous studies have shown that conversion of PtdIns into its downstream PtdInsP derivatives is heavily dependent on the subcellular membrane in which it is located. Only when it is present in specific membranes is it readily available as a substrate for the various specific phosphoinositide kinase and phosphatase enzymes for conversion into a specific PtdInsP. To reach these distinct cellular membranes, PtdIns could be delivered via membrane trafficking events or through the action of the specific lipid-transfer activity of SEC14-like PITPs. These new results suggest that, in addition to PITPs and specific kinase and phosphatase activities, the ability of PtdIns to be converted into downstream PtdInsPs may also depend upon the specific PIS isoform from which it is derived.

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