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346 Biochemical Society Transactions (2006) Volume 34, part 3 Sec14p-like proteins regulate phosphoinositide homoeostasis and intracellular protein and lipid trafficking in yeast C.J. Mousley, K.R. Tyeryar, M.M. Ryan and V.A. Bankaitis1 Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, U.S.A. Abstract The major PI (phosphatidylinositol)/PC (phosphatidylcholine)-transfer protein in yeast, Sec14p, co-ordinates lipid metabolism with protein transport from the Golgi complex. Yeast also express five additional gene products that share 24–65% primary sequence identity with Sec14p. These Sec14p-like proteins are termed SFH (Sec Fourteen Homologue) proteins, and overexpression of certain individual SFH gene products rescues sec14-1ts -associated growth and secretory defects. SFH proteins are atypical in that these stimulate the transfer of PI, but not PC, between distinct membrane bilayer systems in vitro. Further analysis reveals that SFH proteins functionally interact with the Stt4p phosphoinositide 4-kinase to stimulate PtdIns(4,5)P2 synthesis which in turn activates phospholipase D. Finally, genetic analyses indicate that Sfh5p interfaces with the function of specific subunits of the exocyst complex as well as the yeast SNAP-25 (25 kDa synaptosome-associated protein) homologue, Sec9p. Our current view is that Sfh5p regulates PtdIns(4,5)P2 homoeostasis at the plasma membrane, and that Sec9p responds to that regulation. Thus SFH proteins individually regulate specific aspects of lipid metabolism that couple, with exquisite specificity, with key cellular functions. Introduction PITPs (phosphatidylinositol-transfer proteins) are characterized by their ability to mediate the transfer of phosphatidylinositol or PC (phosphatidylcholine) monomers between membrane bilayers in vitro [1,2]. The major PITP in the yeast Saccharomyces cerevisiae, encoded by the essential SEC14 gene, localizes to the Golgi apparatus where its function is necessary for vesicle-mediated protein transport from this organelle [3,4]. Observations regarding the function of Sec14p in vivo derive from extensive characterization of the conditional sec14-1ts allele and analysis of extragenic loss of function mutations that permit cell viability in the complete absence of essential Sec14p function. This ‘bypass Sec14p’ condition has proven invaluable in increasing our understanding of how Sec14p regulates secretory vesicle biogenesis from the trans-Golgi network. We now know it does so by generating an appropriate pro-secretory lipid environment in those membranes [2,3]. Accordingly, Sec14p dysfunction leads to a significant derangement of several aspects of lipid metabolism which subsequently impose a defect in secretory vesicle biogenesis from trans-Golgi membranes Key words: lipid trafficking, phosphatidylinositol-transfer protein (PITP), phospholipase D, Sec14p, target membrane soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (t-SNARE), yeast. Abbreviations used: DAG, diacylglycerol; ER, endoplasmic reticulum; GFP, green fluorescent protein; PA, phosphatidic acid; PI, phosphatidylinositol; PITP, PI-transfer protein; PC, phosphatidylcholine; PLD, phospholipase D; PS, phosphatidylserine; SFH, Sec Fourteen Homologue; (t-)SNARE, (target membrane) soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor. 1 To whom correspondence should be addressed (email [email protected]). C 2006 Biochemical Society [4–9]. This is accompanied by a dramatic disorganization of the actin cytoskeleton in Sec14p-deficient cells [10]. Interestingly, Sec14p-independent cell growth in all ‘bypass Sec14p’ mutants requires the constitutive activation of PLD (phospholipase D) [11,12], encoded by the nonessential SPO14 gene, which catalyses the hydrolysis of PC to choline and PA (phosphatidic acid). It is postulated that the hydrolysis of PC by PLD generates a critical pool of PA that is subsequently converted into DAG (diacylglycerol), and that it is this generation of DAG at the expense of PC that helps compensate for the loss of Sec14p function in sec14-1ts cells [7,12–14]. The essential function of Sec14p notwithstanding, it is now clear the PITP repertoire of even simple yeast cells is more complex than expected. The yeast genome encodes five additional genes whose translational products are homologous with Sec14p and are required for PLD activation in bypass sec14 mutants [12]. Herein, we summarize our current understanding of the function of these Sec14p-like yeast PITPs, and discuss roles for such proteins in regulating membrane trafficking and dynamics of the actin cytoskeleton. The yeast Sec14p-like family The S. cerevisiae genome encodes five genes whose protein products share at least 25% identity and 45% similarity to Sec14p, and are termed SFH1–SFH5 (Sec Fourteen Homologue 1–5) (Figure 1) [12]. Soluble fractions prepared by salt-stripping of membranes harvested from yeast cells overproducing Sfh2p, Sfh3p, Sfh4p or Sfh5p exhibit Non-Vesicular Intracellular Traffic Figure 1 The S. cerevisiae Sec14p-like gene family A schematic comparison of Sec14p with the five yeast Sec14p-like proteins is shown. The gene names are given on the left and the percentage primary sequence identity/similarity to Sec14p is indicated within the grey area that represents the region of homology with Sec14p. The number of residues in each protein is given at the upper right of each corresponding box depiction. Sec14p-independent PI (phosphatidylinositol)-transfer activity in vitro, indicating that the corresponding SFH proteins are also PITPs. These SFH proteins are biochemically distinguished from Sec14p in their inability to transfer PC in vitro, however [12]. That Sfh2p, Sfh4p and Sfh5p are functionally related to Sec14p is indicated by two additional lines of evidence. Firstly, increased gene dosage of SFH2, SFH4 or SFH5 is sufficient to rescue the conditional lethality of sec14-1ts yeast. Secondly, this phenotypic rescue is accompanied by improved Golgi secretory capacity, as demonstrated by the increased capacity of sec14-1ts cells to secrete invertase [12]. It is also interesting that the SFH protein most related to Sec14p by homology is not a functional Sec14p on the basis of several important criteria. Yeast cells overexpressing Sfh1p have at best a very low grade Sec14p-independent PI-transfer activity in vitro, and Sfh1p overexpression does not effect an efficient rescue of sec14 defects in vivo [12,15]. Their biochemical similarities notwithstanding, we now know that SFH proteins represent a novel class of nonclassical fungal PITPs whose individual functions are substantially unique and non-redundant. The nature of these individual functions is only now coming to light, and recent progress on this subject is reviewed below. SFH PITPs and Stt4p stimulate PLD activity The SFH proteins do not individually, or collectively, execute essential cellular functions. Also, en bloc deletion of the SFH genes in the sec14-1ts genetic background does not compromise cell viability, implying that SFH proteins do not define a minor family of PITPs which share functional redundancy with Sec14p. However, SFH proteins are required for Sec14p-independent cell growth in ‘bypass Sec14p’ mutants, as collective deletion of all SFH genes abolishes Sec14pindependent growth [12]. Intriguingly, this phenotype is reminiscent of that associated with PLD deficiency in sec14-1ts cells, as constitutive PLD activity is required to support Sec14p-independent cell growth in all ‘bypass Sec14p’ mutants [11]. The SFH/PLD phenotypic similarity in the context of ‘bypass Sec14p’ suggested a first clue regarding a biological function for the SFH PITPs, namely, a role for SFH proteins in optimal stimulation of PLD activity. Experimental evidence supports this concept. Collective ablation of the SFH2– SFH5 genes in yeast significantly compromises the PLD activity in vivo evoked by Sec14p inactivation, as measured both by PLD-dependent release of choline and generation of PA [12]. Thus SFH proteins support the course of PLD activation that occurs in Sec14p-deficient cells. This finding enlightens our understanding of a conundrum presented by the demonstration that PLD activity is strongly elevated upon inactivation of Sec14p. Liscovitch and Cantley [16] have previously synthesized the known lipid activation properties of PLD and phosphoinositide kinases into an attractive proposal that PITPs drive a positive feedback loop that stimulates PLD enzymatic activity. While the activation of PLD by inactivation of Sec14p is spectacularly counter to this model, the potential employment of the SFH PITPs in an SFH/phosphoinositide kinase/PLD regulatory circuit provides an alternative PITP/PLD circuit that satisfies the basic tenets of the same model. SFH proteins regulate PI metabolism in vivo The biochemical properties of SFH proteins suggest a simple mechanism for how these PITPs activate PLD. Given that PtdIns(4,5)P2 is an obligate cofactor for PLD enzymatic activity [17], we anticipated that SFH proteins stimulate the synthesis of 4-OH phosphorylated phosphoinositides in vivo. Indeed, SFH2 overexpression increases both PtdIns(4)P and PtdIns(4,5)P2 in yeast strains with baseline phosphoinositide levels, yet PtdIns(3)P levels are not affected [10]. Overproduction of Sfh4p and Sfh5p, unlike overproduction of Sfh2p, does not increase PtdIns(4)P, but does result in increased PtdIns(4,5)P2 . Taken together, these findings imply that Sfh2p, Sfh4p and Sfh5p modulate PtdIns(4)P and/or PtdIns(4,5)P2 metabolism in vivo. Similarly, collective ablation of the SFH2–SFH5 genes evokes a 40% reduction in bulk PtdIns(4,5)P2 relative to isogenic wild-type controls [10]. How do these non-conventional PITPs stimulate phosphoinositide synthesis? Sfh2p, Sfh4p and Sfh5p do not appear to directly stimulate the activity of any of the three yeast phosphoinositide 4-kinases (e.g. Pik1p, Stt4p and Lsb6p), nor do they stimulate the activity of the single yeast phosphoinositide 4-phosphate 5-kinase in vitro (Mss4p; [10]). Rather, SFH proteins probably stimulate phosphoinositide production by regulating the delivery of PI substrate to the appropriate phosphoinositide kinase(s). Do SFH proteins channel PI to a specific phosphoinositide kinase or are these proteins more promiscuous in this regard in the usual physiological setting? The results indicate that the functional SFH/phosphoinositide kinase interface is a C 2006 Biochemical Society 347 348 Biochemical Society Transactions (2006) Volume 34, part 3 Table 1 Increased phosphatidylinositol 4-phosphate in phosphoinositide phosphatase-deficient yeast with elevated Sfh2p or Sfh5p Yeast sec14∆ cki1∆ double mutants exhibit baseline levels of all phosphoinositide species and these mutants are specifically sensitized to increased Stt4p activity by inactivation of the Sac1p phosphoinositide phosphatase [10]. Steady-state phosphatidylinositol 4-phosphate levels for the parental triple mutant strain carrying a control YEp(URA3) plasmid or Sfh2p and Sfh5p overexpression plasmids (YEpSFH2 and YEpSFH5 respectively) are given. Values are expressed as percentage of total deacylatable inositol phospholipids. Phosphatidylinositol 4-phosphate Yeast strain genotype (% total deacylatable inositol phospholipid) sec14∆ cki1 sac1∆ YEp(URA3) sec14∆ cki1 sac1∆ YEp(SFH2) sec14∆ cki1 sac1∆ YEp(SFH5) 5.7 ± 0.4 7.6 ± 0.9 9.3 ± 0.5 specific one. Disruption of either SFH2 or SFH5 exacerbates the growth defects associated with stt4-4ts , but not pik1-83ts growth defects, implying that both Sfh2p and Sfh5p functionally interact with Stt4p [10]. In agreement with the genetic interaction data, Stt4p-dependent PtdIns(4)P synthesis is stimulated by the overexpression of either SFH2 or SFH5 when the experiment is performed in a phosphoinositide phosphatase-deficient genetic background (Table 1) [10]. A functional interface between the Sfh proteins and Stt4p strongly implies that these molecules are important in synthesizing extra-Golgi pools of phosphoinositides. As specific SFH proteins functionally interact with Stt4p, and SFH proteins are required to activate PLD, it is expected that PLD activity should also be sensitive to robustness of Stt4p function. This expectation is also realized as thermal inactivation of the Stt4-4ts protein evokes reductions in PLD activation that are comparable in magnitude with those recorded upon wholesale deletion of the SFH genes [10]. This is fully consistent with the idea that PLD activation in vegetative cells requires the concerted activity of SFH proteins and Stt4p. Sfh2p and Sfh5p restore polarized actin organization to Sec14p-deficient cells Are there other downstream cellular effects mediated by SFH regulation of the available PtdIns(4,5)P2 pool? In this regard, Sec14p orthologues in Schizosaccharomyces pombe and Arabidopsis thaliana are implicated in modulating postGolgi membrane trafficking as well as in the organization of the actin cytoskeleton [18,19]. Actin polarization is responsive to exocytic membrane flux and phosphoinositides [20,21]. In yeast, the distribution of actin is tightly polarized whereby actin filaments traverse the entire length of the mother cell, enter the bud, and concentrate as cortical patches within the bud. However, this distinct organization is dramatically compromised in Sec14p-deficient yeast as the cortical C 2006 Biochemical Society actin patches become distributed throughout the mother and daughter cells as puncta and the actin cables are diminished [10]. Consistent with SFH proteins displaying functional homology with Sec14p, overexpression of SFH2 or SFH5 corrects the actin cytoskeletal disorganization that stems from Sec14p insufficiency. The effects of SFH proteins on regulation of actin dynamics are also discernible in SEC14 yeast. The actin cytoskeleton in sfh∆ mutants is unable to undergo the rapid remodelling normally recorded when yeast cells are challenged with stresses such as heat shock [10]. Sfh5p-mediated control of plasma membrane PtdIns(4,5)P2 and efficient Sec9p t-SNARE (target membrane soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) function Stt4p and Mss4p reside in the yeast plasma membrane [22]. This raises the possibility that SFH proteins modulate exocytic and/or endocytic events at the plasma membrane. To investigate whether the yeast SFH proteins modulate exocytosis, each SFH gene product was individually overexpressed in a number of sec ts mutants that block membrane trafficking at various points along the secretory pathway. Overexpression of SFH1–SFH4 fails to restore growth of any sects strain (other than sec14ts ) at restrictive temperatures, but SFH5 overexpression reverses the temperature-sensitive growth and secretory defects associated with each of the sec8-9ts , sec10-2ts and sec15-1ts mutations [10]. These mutations all affect subunits of the exocyst complex that itself plays an essential role in docking the 90 nm Golgi-derived secretory vesicles to the plasma membrane [23]. The mechanism by which overexpression of SFH5 suppresses defects in components of the exocyst is PLD-independent. How does Sfh5p levy these effects on exocyst function? The answer seems to lie in the interface between regulation of plasma membrane PtdIns(4,5)P2 levels and the activity of Sec9p, the yeast version of the neuronal SNAP-25 (25 kDa synaptosome-associated protein) [24]. In this regard, overexpression of the Mss4p phosphoinositide 4-phosphate 5kinase resembles Sfh5p overexpression in its partial rescue of the temperature-sensitive growth defects associated with sec8-9ts , sec10-2ts and sec15-1ts mutant alleles, as well as growth defects associated with sec9ts , sec1ts and sec2ts mutants. By the same token, overexpression of the Sec9p t-SNARE elicits partial rescue of a spectrum of sects mutations that very closely resembles that recorded for Mss4p overexpression [10]. Both the Mss4p- and the Sec9p-dependent rescue is Sfh5p-dependent and PLD-independent. Moreover, the Mss4p-dependent rescue is specific with respect to the phosphoinositide-4-phosphate 5-kinase activity, as elevated dosage of either the PIK1 or LSB6 phosphoinositide 4-kinase structural genes fails to evoke the same effects [10]. The Sfh5p-dependent rescue of post-Golgi sects secretory defects levied by overexpression of Mss4p implies a mechanism involving elevation of a plasma membrane pool of PtdIns(4,5)P2 , and evidence in favour has been obtained Non-Vesicular Intracellular Traffic Figure 2 Sfh5p localizes to peripheral ER Figure 3 An intermembrane contact site model for SFH protein Fluorescence imaging experiments show that a Myc epitope-tagged Sfh5p (in red; upper right) substantially co-localizes with the peripheral, function (A) An SFH protein (grey) may itself be an integral component of an but not the nuclear envelope-associated, aspect of the ER, as marked by a Sec61p–GFP chimaera (in green; upper left). The merge panel (lower right) shows that Sfh5p is restricted to discrete subdomains of peripheral intermembrane contact site and employ its PI-binding capacity (in black) to impose specific PI trafficking through that site. (B) An SFH protein may employ its ability to channel PI to a phosphoinositide kinase to generate ER (arrows). That Sfh5p is not co-localized with Stt4p on the yeast plasma membrane was demonstrated previously [10]. The lower left panel shows a Nomarski image of the cells examined. a phosphoinositide platform that recruits intrinsic components (Factor X) of an intermembrane contact site. In this scenario, the SFH protein does not directly utilize its PI-binding properties to impose PI-trafficking specificity through such a site. Other lipid species could pass through such a site. from PtdIns(4,5)P2 imaging experiments. A PLD–GFP (green fluorescent protein) chimaera is a reliable PtdIns(4,5)P2 sensor that translocates to the plasma membrane upon Mss4p overexpression [25]. This relocalization of PLD–GFP is compromised in sfh5∆ cells, indicating that Sfh5p contributes to the synthesis and/or maintenance of the Mss4p-dependent pool of plasma membrane PtdIns(4,5)P2 [10]. How does PtdIns(4,5)P2 regulate Sec9p t-SNARE function? While the details are unclear, phosphoinositidemediated regulation of SNARE function may well emerge as a common theme. In this regard, PtdIns(4,5)P2 reduces the diffusion rate of t-SNAREs in membranes [26]. Also, phosphoinositides co-operate with DAG and ergosterol in controlling the formation of SNARE complexes required for the homotypic fusion of vacuolar membranes [27]. Discussion Herein, we discuss the body of biochemical and genetic evidence to indicate that SFH proteins are non-classical PITPs that channel PI to the Stt4p phosphoinositide 4-kinase. This metabolic channelling influences a number of cellular functions that include PLD activation, organization of the cytoskeleton and, in the case of Sfh5p, plasma membrane phosphoinositide homoeostasis. In the last case, the Sfh5presponsive plasma membrane PtdIns(4,5)P2 pool couples with the activity of the post-Golgi secretory vesicle docking/ fusion machinery at the plasma membrane. Our interpreta- tion of the various results presented here (and elsewhere) is that SFH proteins, and PITPs in general, are individually dedicated to the modulation of specific lipid metabolic events that themselves couple with very specific biological functions. This exquisite functional specificity is at odds with the startling functional promiscuity exhibited by PITPs in cell-free or permeabilized membrane systems that purportedly reconstitute PITP-dependent processes. We find it an attractive notion that SFH proteins, and perhaps PITPs in general, represent specificity factors that help define the identity of distinct phosphoinositide/lipid pools. How SFH proteins may play a role in PI channelling and pool specification is unclear. With regard to channelling, SFH proteins are peripheral membrane proteins that need to be salt-stripped from membranes [10]. Thus these proteins may not mobilize PI between membranes via a soluble PI/SFH protein transport intermediate. The case of Sfh5p suggests other interesting possibilities. Sfh5p, while functionally engaged with Stt4p, nonetheless localizes to subdomains of the peripheral ER (endoplasmic reticulum) (Figure 2), and not to the plasma membrane where Stt4p resides. The localization patterns of Sfh5p and Stt4p are not coincident [10]. Taken together, the results are consistent with a model whereby Sfh5p acts as a component of a machinery that forms intermembrane contact sites, thereby providing a nonvesicular and non-carrier mechanism for transfer of PI from one membrane to another (Figure 3). The lipid-binding specificity of Sfh5p could either gate the contact site with regard to which lipids are competent to pass through it, or Sfh5p may function in regulating the assembly of such a site. C 2006 Biochemical Society 349 350 Biochemical Society Transactions (2006) Volume 34, part 3 The contact site model may be generally applicable to the SFH proteins. In this regard, Sfh4p is implicated in an intermembrane contact site-dependent transport of PS (phosphatidylserine) from the ER to the extramitochondrial PS decarboxylase [28] where it is subsequently converted into phosphatidylethanolamine. Interestingly, this metabolic involvement is specific to Sfh4p, as neither Sec14p, Sfh2p, Sfh3p nor Sfh5p can substitute for Sfh4p in this pathway [10]. This finding argues that SFH proteins do not define general components of a putative contact site machinery. 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