Yeast Sec14p acts as a phosphatidylinositol/phosphatidylcholine-transfer protein in vitro. In vivo, it is essential in promoting Golgi secretory function. Products of five genes named SFH1–SFH5 (Sec Fourteen Homologues 1–5) exhibit significant sequence homology to Sec14p and together they form the Sec14p family of lipid-transfer proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism and membrane trafficking.
- Golgi secretion
- lipid metabolism
- phosphatidylinositol-transfer protein
- Sec14 homologue
Eukaryotic cells contain highly specialized subcellular compartments with very elaborate and specific lipid composition of their membranes. Regulation of biological membrane lipid composition is extremely complex; it includes co-ordination of synthesis, transport, remodelling and degradation of lipids. Lipids are not only a subject of this regulation but also active participants in many important sensing and signalling pathways. The focus of the present paper is the role of the yeast Saccharomyces cerevisiae PI (phosphatidylinositol)/PC (phosphatidylcholine)-transfer protein, Sec14p, and its homologues in modulation of membrane lipid composition.
Yeast PI/PC-transfer protein, Sec14p
A phospholipid-transfer protein that facilitated the transfer of PI and PC in vitro between biological and artificial membranes was isolated from yeast cytosol and purified to homogeneity . Later it was shown that this protein is encoded by the SEC14 gene, it is required for transport of secretory proteins from the yeast Golgi complex and it is essential for cell viability [2,3]. Importantly, PITPs (PI-transfer proteins) were found in all eukaryotes examined so far and their significance is evident from the studies in mice and flies, where insufficiencies in PITPs lead to severe neurodegenerative defects [4–6]. However, it should be noted here that mammalian PITPs are similar to S. cerevisiae Sec14p in size, lipid binding and transfer properties, but have no sequence homology to Sec14p. Functionally, mammalian and yeast PITPs partially overlap [7,8].
In vitro PI/PC-transfer activity of Sec14p suggests its role in the intracellular trafficking of phospholipids. It has become clear that Sec14p fulfils an important role at the interface of lipid metabolism with membrane trafficking, but much of the molecular details remain unknown. The current view is that Sec14p is not a passive mediator of phospholipid transfer but regulates in a complex way phospholipid metabolic pathways that have an impact on signalling and membrane traffic.
The first clues about the in vivo role of Sec14p came from the analysis of genetic suppressors of the essential Sec14p requirement for Golgi function and cell viability. It was found that inactivation of any one of the structural genes encoding enzymes of the CDP-choline (or Kennedy) pathway for PC biosynthesis ‘bypass’ the requirement of Sec14p for secretion and viability . Connection between the function of Sec14p and metabolism of the major membrane phospholipid, PC, was further strengthened by the finding that PC-bound Sec14p down-regulates a key enzyme of the CDP-choline pathway, choline phosphate cytidylyltransferase . Inactivation of the second pathway leading to PC biosynthesis, the PE (phosphatidylethanolamine) pathway, together with inhibition of choline uptake from the medium, also rescue the growth and secretory defects associated with Sec14p deficiency . Another key observation was that intact PLD1 (phospholipase D1; encoded by the SPO14 gene) must be present for all genetic suppressors (not only for those directly related to PC biosynthesis) to bypass the Sec14p requirement [12,13]. PLD1 releases choline from the PC molecule and produces one molecule of PA (phosphatidic acid) for every molecule of PC degraded. In addition, it was shown very recently that Sec14p formally behaves as a positive regulator of Nte1p (intracellular PLB)-mediated PC degradation, resulting in formation of free (unbound) fatty acids and glycerophosphocholine . Taken together, this evidence indicates that one of the roles of Sec14p is to maintain PC levels of the Golgi membranes at the level compatible with generation of secretory vesicles and/or to regulate the amount of PC degradation products, PA or DAG (diacylglycerol) required for secretion to proceed.
Another important suppressor of Sec14p requirement in Golgi secretion and cell viability is sac1 . SAC1 was found to encode a PI 4-phosphatase . Corresponding with this activity, Stock et al.  found highly elevated PtdIns(4)P levels in the sec14 sac1 double mutant when compared with the sec14 mutant. Importantly, later experiments revealed that PtdIns(4)P levels were significantly decreased in the mutant with temperature-sensitive sec14-1 allele when shifted to a non-permissive temperature of 37 °C . In addition, specific overexpression of PIK1, encoding one of two essential phosphoinositide 4-kinases, was able to partially rescue sec14-1 cells at normally non-permissive temperature. These findings indicate that PtdIns(4)P deficiency is a major factor contributing to the secretory defect in sec14 cells and that PtdIns(4)P has an important role in the Golgi secretion. A still unresolved question is whether elevated levels of PtdIns(4)P in the sec14 sac1 strain directly facilitate secretory compatibility in the absence of functional Sec14p or if increased levels of PtdIns(4)P play an indirect role by altering phospholipid metabolism.
The final gene whose inactivation resulted in the ability of yeast to survive in the absence of Sec14p is the KES1 gene, encoding an oxysterol-binding protein family member . It was proposed that Kes1p function interfaces with the activity of the yeast Arf (ADP-ribosylation factor) cycle . Kes1p is a peripheral Golgi membrane protein whose correct localization to the Golgi apparatus requires PtdIns(4)P binding. This Kes1p feature links the yeast Arf cycle with Golgi apparatus levels of phosphoinositides . Analysis of genetic interactions with Kes1p helped to identify the downstream effectors that respond to Sec14p-mediated regulation of lipid metabolism, two ArfGAPs (Arf GTPase-activating proteins), Gcs1p and Age2p [20,21]. Gcs1p and Age2p form an essential ArfGAP pair that provides overlapping function for transport from the Golgi apparatus . Mutant cells lacking both of these proteins display defects strongly resembling those of sec14 mutants, namely loss of cell viability, accumulation of membranous structures resembling Berkeley bodies and blockage in the secretory pathway. Notably, both of these ArfGAP activities are stimulated by DAG and PA, and inhibited by PC , providing a connection between the Sec14p-regulated membrane lipid composition and the components of the secretory machinery.
In summary, the activity of Sec14p helps to establish the Golgi membrane lipid composition that is optimal for secretion from the trans-Golgi network, mediated by two ArfGAP proteins Gcs1 and Age2 (Scheme 1). In the absence of a functional Sec14p alternative, ‘bypass’ mechanisms create a membrane lipid environment compatible with the secretory process (Scheme 1). As a result of these ‘bypass’ mechanisms, (i) decrease in PC synthesis, (ii) increased PC degradation accompanied by increase in PA/DAG production, and (iii) increase in PI and PtdIns(4)P levels were observed. One must, however, exercise care when directly connecting these ‘bypass’ mechanisms to the function of Sec14p because these ‘bypass’ mechanisms do not completely recapitulate the Sec14p-mediated functions. For example, one of the Sec14 bypass strains, sec14 cki1 mutant (CKI1 gene encodes choline kinase in the CDP-choline pathway for PC biosynthesis), is viable, but the secretion pattern of the periplasmic protein invertase is significantly different from the control strain having functional Sec14p . In addition, in the sec14 cki1 mutant, phospholipid biosynthesis is deregulated on the transcriptional level [23,24].
Sec14p homologues in yeast
Another way to make Sec14p-deficient yeast viable and secretory-competent is to overexpress the SFH2 (Sec Fourteen Homologue 2) or SFH4 genes (and to a much lesser degree SFH1 or SFH5), encoding the members of Sec14 group of homologous proteins in S. cerevisiae [25,26]. The yeast genome contains five genes named SFH1–SFH5 whose products exhibit significant sequence homology to Sec14p (Figure 1). Initial characterization of these Sec14 homologues revealed that all but one of them (Sfh1p) are PITPs exhibiting PI- but not PC-transfer activity. Sfh1p, despite its high sequence homology to Sec14p, is neither a PI- nor a PC-transfer protein in vitro . None of the five Sec14 homologues individually is essential for growth under standard conditions; however, they are collectively required for the Sec14p-independent cell growth and for the optimal activation of PLD1 in Sec14p-deficient cells [25,26].
Sfh1p is the most homologous protein to Sec14p (Figure 1). This protein conserves all recognized critical structural motifs of Sec14p . Therefore it was surprising that Sfh1p does not possess either PI- or PC-transfer activity in vitro and that, if overexpressed, it complements the Sec14p-related functions only to a very limited degree [25,26]. We hypothesize that the reason for this weak growth complementation of Sec14p deficiency is that Sfh1p is localized to the nucleus and Sec14p is predominantly a cytosolic protein . Interestingly, subcellular localization of yeast proteins, Sec14p (mostly cytosolic with Golgi function) and Sfh1p (predominantly nuclear), resembles the localization of PITP isoforms in higher eukaryotes; PITP-α being predominantly present in the nucleus and cytoplasm and PITP-β in the cytoplasm and Golgi apparatus .
Sfh2 protein, despite its modest identity (28%) to Sec14p, seems to have several functions in common with Sec14p. When overexpressed, it effectively complements the growth and secretion defect associated with the non-functional Sec14p [25,26]. For this complementation, it does not require functional PLD. Sfh2p is involved in modulation of PC degradation pathways via PLB and PLD . Two recent papers put Sfh2p directly at the scene of Golgi secretion. The first one by Wong et al.  provides evidence that ArfGAP Gcs1p is subject to temporal and spatial regulation facilitated by Sfh2p-mediated modulation of the lipid environment. The other one by Routt et al.  shows that Sfh2p stimulates phosphoinositide synthesis.
The SFH3 gene was first identified in the genomic screen for the genes that are regulated by the multiple drug resistance regulator Pdr1p and named accordingly PDR16 . Deletion of the PDR16/SFH3 gene resulted in significant changes in sterol composition and hypersensitivity of yeast to azole inhibitors of ergosterol biosynthesis. Overexpression of the SFH3 gene does not complement the non-functional or missing Sec14p. Currently, we can only speculate that Sfh3p could play a role in trafficking of ergosterol, its precursors, or esters based on the dual localization of the Sfh3p to lipid particles and cell periphery . Yeast lipid particles are the site of sterol storage in the form of sterol esters and the plasma membrane is the cellular membrane most abundant in sterols [32,33].
Sfh4p was identified as an essential component of the transport machinery required for PS (phosphatidylserine) delivery to the Golgi apparatus . In the Golgi apparatus, PS can be converted into PE by the enzymatic activity of Psd2p (PS decarboxylase 2) . Sfh4p (in the papers dealing with PS transport in yeast named PstB2p) was found in either soluble form or membrane-bound on the acceptor membrane . This protein forms an essential part of the docking/transport complex bridging the narrow gap between the endoplasmic reticulum membrane and the Golgi apparatus, allowing the transfer of PS to the location of Psd2p. As with other yeast PITPs, a clear connection to the metabolism of phosphoinositides and PA was observed for Sfh4p. First, an important part of the above-mentioned docking/transport machinery is the phosphoinositide 4-kinase, Stt4p. Secondly, it was demonstrated in PS transport reconstitution experiments that PA and PtdIns(4)P increased transport from chemically defined donor vesicles made of PS and other lipids . An exciting possibility is that the role of Sfh4p in vesicle-independent lipid transport could reflect a much broader aspect of PITPs physiology – to mediate events at the membrane contact sites. Such a mechanism of action was recently proposed by Holthuis and Levine .
Sfh5p complements growth and secretion defects associated with the non-functional Sec14p only to a limited degree when overexpressed from the powerful promoter of the yeast phosphoglycerate kinase gene . Recently, it was shown that this protein regulates PLD1 activity by stimulation of PtdIns(4,5)P2 synthesis and functions at late stages of exocytosis in yeast .
Sec14p deficient in lipid transfer
The yeast PITP, Sec14p, binds and transfers two phospholipids, PI and PC, in an exchange reaction between membranes in vitro. An important question in deciphering the molecular mechanisms by which Sec14p executes its biological functions is to understand the physiological role of each individual phospholipid-binding/transfer activity of Sec14p. Availability of the crystal structure of Sec14p  allowed Phillips et al.  to generate a double mutant Sec14pK66A/K239A deficient in PI-transfer activity but capable of PC-transfer activity in vitro. Surprisingly, expression of physiological levels of this mutant Sec14pK66A/K239A rescued the lethality and Golgi secretory defects associated with sec14 null mutation. This observation suggests that Sec14p does not serve as phospholipid exchange protein between membranes in living cells. What is then the relevance of PC binding and/or transfer in vitro to the physiological role of Sec14p in the cell? To address this question, we attempted to generate a mutated Sec14p unable to transfer PC between membranes in vitro. Based on our previous experience [23,24], we anticipated that such a mutant Sec14p would not be able to properly regulate phospholipid biosynthesis on the transcriptional level. Using a random in vitro mutagenesis approach and a genetic screen based on complementation of the defect in regulation of phospholipid biosynthesis, we identified a Sec14p mutant unable to transfer PC between liposomes in vitro (D. Tahotna and R. Holic, unpublished work). This Sec14p mutant was, similar to PI-transfer-deficient mutant Sec14pK66A/K239A, able to rescue efficiently the lethality and secretion defects associated with sec14 null mutation. The dispensability of PC-transfer activity for the essential in vivo function of Sec14p is further supported by the observation that two Sec14p homologues, Sfh2p and Sfh4p, that do not possess PC-transfer activity  are able to substitute for Sec14p [25,26].
Clearly, proteins of the yeast Sec14 group fulfil specific roles at the interface between lipid metabolism and important cellular functions. Experimental evidence indicates that the role of yeast PITPs goes beyond the in vitro observed phospholipid-transfer activities. Rather, it includes complex regulation of PC, PA, DAG, PI and PtdIns(4)P metabolism that form an appropriate lipid environment for essential trafficking processes to proceed. Despite enormous progress made in recent years, some fundamental questions remain to be addressed. One of them is what physiological function(s) does PITP binding and/or transfer of PI and PC play in cellular physiology. The availability of Sec14p mutants defective in individual transfer activities will certainly advance our knowledge of the mechanisms by which Sec14p and its homologues execute their biological functions.
Critical reading of this paper and comments of Jana Patton-Vogt (Duquesne University, Pittsburgh, PA, U.S.A.) are greatly appreciated. The work in our laboratory was supported by VEGA 2/4130/24 and Science and Technology Assistance Agency APVT-51-024904 grants.
Non-Vesicular Intracellular Traffic: Biochemical Society Focused Meeting held at Goodenough College, London, U.K., 15–16 December 2005. Organized and edited by S. Cockcroft (University College London, U.K.) and T. Levine (Institute of Ophthalmology, London, U.K.).
Abbreviations: Arf, ADP-ribosylation factor; ArfGAP, Arf GTPase-activating protein; DAG, diacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PITP, PI-transfer protein; PLD1, phospholipase D1; PS, phosphatidylserine; Psd2p, PS decarboxylase 2
- © 2006 The Biochemical Society