© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/tra.12236
Review
Transport of Phosphatidylserine from the Endoplasmic Reticulum to the Site of Phosphatidylserine Decarboxylase2 in Yeast Muthukumar Kannan1 , Wayne R. Riekhof2,∗ and Dennis R. Voelker1,∗ 1 Department 2 School ∗
of Medicine and Program in Cell Biology, National Jewish Health, Denver, CO 80206, USA of Biological Sciences, University of Nebraska, Lincoln, NE 68588, USA
Corresponding authors: Dennis R. Voelker, [email protected] and Wayne R. Riekhof, [email protected]
Abstract Over the past two decades, most of the genes specifying lipid synthesis
non-catalytic N-terminal domains act as a hub to nucleate the assembly
and metabolism in yeast have been identified and characterized. Several
of a multiprotein complex, which facilitates PtdSer transport at mem-
of these biosynthetic genes and their encoded enzymes have provided
brane contact sites between the ER and Golgi/endosome membranes.
valuable tools for the genetic and biochemical dissection of interor-
After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to
ganelle lipid transport processes in yeast. One such pathway involves
form PtdEtn, which is disseminated throughout the cell to support the
the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticu-
structural and functional needs of multiple membranes.
lum (ER), and its non-vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine
Keywords endoplasmic reticulum, endosomes, Golgi apparatus, lipid transport, phosphatidylethanolamine, phosphatidylserine decarboxylases
their role in studies of the transport-dependent pathways of de novo syn-
Received 23 September 2014, revised and accepted for publication 28
thesis of phosphatidylethanolamine (PtdEtn). The emerging picture of
October 2014, uncorrected manuscript published online 30 October
the Psd2p-specific transport pathway is one in which the enzyme and its
2014, published online 11 December 2014
Identification of Psd1p, Psd2p and Their Application as Reporters of PtdSer Traffic
as the major sites of glycerophospholipid biosynthesis in eukaryotes (9–11). In yeast, a significant proportion of total PtdEtn can also be synthesized in the compartments of the Golgi and endosomes (12,13). The compartmentation of lipid-synthesizing activities necessitates efficient interorganelle transport to membranes incapable of autonomous lipid synthesis, in order to maintain membrane biogenesis and function.
Phosphatidylethanolamine (PtdEtn) is an abundant membrane phospholipid of yeast as well as many lower and higher eukaryotes and bacteria. In addition to acting as a structural constituent of membranes, PtdEtn is involved in numerous specific cellular functions including autophagy (1,2), fusion and fission events of membranes (3), vacuolar protein delivery (4), the stabilization of membrane proteins or enzymes (5,6) and cell signaling (7,8). The endoplasmic reticulum (ER) and mitochondria are generally accepted
The topological segregation of yeast enzymes involved in aminoglycerophospholipid synthesis is shown in Figure 1. In this scheme, phosphatidylserine synthase (Pss1p, encoded by the CHO1/PSS1 gene) (14) is localized in the www.traffic.dk 123
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exported to other membranes to fulfill their requirements for PtdEtn (23). Many eukaryotes, including yeast, can efficiently methylate PtdEtn to form phosphatidylcholine (PtdCho) in the ER (24).
Figure 1: Topology of de novo PtdCho and PtdEtn biosynthesis in yeast. Major pathways of aminoglycerophospholipid synthesis are depicted, with an emphasis on transport-dependent enzymatic reactions. Interorganelle transport events are denoted with dashed lines, and enzymes are denoted with the standard yeast genetic nomenclature. Pss1p, phosphatidylserine synthase; Psd1p, phosphatidylserine decarboxylase 1; Psd2p, phosphatidylserine decarboxylase2; Pem1p, phosphatidylethanolamine methyltransferase 1; Pem2p, phosphatidylethanolamine methyltransferase 2. The Kennedy pathways constitute reactions for the synthesis of PtdEtn and PtdCho in the ER that proceed through phospho-ethanolamine/choline and CDP-ethanolamine/choline intermediates. ER and mitochondria-associated ER membranes (MAMs) (15,16). Phosphatidylserine decarboxylase1 (Psd1p) is present in mitochondria (17), and phosphatidylserine decarboxylase2 (Psd2p) is distributed in the Golgi and endosomes (12,13,18). Phosphatidylethanolamine methyltransferases 1 and 2 (encoded by the CHO2/PEM1 and OPI3/PEM2 genes) reside in the ER (16), as corroborated by green fluorescent protein (GFP) localization studies (19). Following the synthesis of phosphatidylserine (PtdSer) in the ER, this lipid must be transported to either mitochondria or Golgi/endosome compartments for decarboxylation to generate PtdEtn. The PtdEtn produced within the mitochondria is required for the optimal function of this organelle (17,20), and yeast strains lacking Psd1p undergo conversion to petite phenotypes with high frequency. PtdEtn produced in the ER by the CDP-ethanolamine (Etn)-dependent ‘Kennedy’ pathway (21) is only poorly transported into mitochondria, and cannot fully restore all functions of the pool produced within the mitochondria (12,17,20,22). PtdEtn synthesized by Psd1p within the mitochondria is not static, and can be 124
The Golgi apparatus also has a limited capacity to synthesize some glycerophospholipids (25,26). In yeast, PtdSer transported to the Golgi can also be a substrate for Psd2p (12). The flux of PtdSer through the Psd2p enzyme accounts for up to 30% of the total de novo biosynthetic flux (13), when the Psd1p enzyme is also active, and the resultant PtdEtn can fulfill most cellular needs for this lipid. However, similar to the Kennedy pathway-derived PtdEtn pool, PtdEtn derived from Psd2p is insufficient to completely provide the PtdEtn pool required by mitochondria under respiratory conditions (20,22). When required, the PtdEtn produced by Psd2p in the Golgi/endosome can be transported to the ER for methylation to PtdCho, and serve to fulfill all the cellular needs for these two lipids under non-respiratory conditions (e.g. glucose as carbon source) (12,18).
Biochemical Function of Phosphatidylserine Decarboxylases PSD enzymes play a central role in the phospholipid metabolism of organisms ranging from bacteria to humans, and a similar enzymatic function is likely to be involved in archaeal ether lipid synthesis (27). As described above and in Figure 1, the yeast Saccharomyces cerevisiae expresses two different PSD activities: one associated with the inner mitochondrial membrane (IMM) (Psd1p) (17), as well as a non-mitochondrial enzyme (Psd2p) associated with the Golgi and endosomes (12,13). The PSD enzymes decarboxylate only the lipid-linked form of the serine moiety. Derivatives of PtdSer, including serine, phosphoserine and glycerophosphoserine, fail to serve as either substrates or inhibitors of the catalytic activity (28). Although no extensive surveys of the fatty acid preference of the purified enzymes have been reported, PSDs will utilize substrates containing saturated and unsaturated fatty acids equally well (29). Synthetic substrates such as dimyristoyl PtdSer, 1-acyl2[N(6-[7-nitrobenz-2-oxa-1,3diazo-4-yl]-aminocaproyl)]PtdSer (NBD-PtdSer) (30) and 1-acyl-2-ω-pyreneacyl-PtdSer (31) are also recognized and decarboxylated Traffic 2015; 16: 123–134
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by the enzyme, suggesting that the minimum requirement for substrate recognition is a glycerophosphoserine moiety to which two hydrophobic substituents are esterified.
Role of PtdSer Decarboxylases in Transport-Dependent Metabolism of Aminoglycerophospholipids The identification and characterization of genes encoding the yeast PSD enzymes provided important tools for the study of aminoglycerophospholipid transport, facilitating genetic studies, in which mutants could be isolated by simple auxotrophic screens. The genetic manipulation (deletion and/or overexpression) of PSD genes in yeast has thus served as a valuable tool for studying phospholipid transport among different organelles (32,33). The pathways for lipid transport have been conceptually divided into two major branches for simplicity. The ‘A’ pathway describes the movement of PtdSer into- and PtdEtn out of the mitochondria. The psd2Δ strains serve as a tool to study ‘A’ pathways by forcing all biosynthetic flux of PtdSer through the Psd1p-dependent branch. Similarly, the ‘B’ pathways use psd1Δ strains to force the PtdSer movement into and PtdEtn transport out of the Golgi/endosome. These complementary approaches have allowed the identification of strains with defects in both ‘A’ and ‘B’ pathways, and facilitated the cloning and characterization of the corresponding mutant genes and their products (34–40).
Important Elements of PSD Structure The primary structures of yeast PSDs comprise 500 amino acids in Psd1p and 1138 amino acids in Psd2p, corresponding to molecular masses of 56.5 and 130 kDa, respectively
(17,18,41,42). The first complete eukaryotic gene encoding a PSD (PSD1) was cloned and sequenced from yeast (17). Deletion of this gene revealed a second, non-mitochondrial activity which was denoted Psd2p, and the cloning and characterization of the PSD2 locus followed, and demonstrated that psd1Δ psd2Δ double mutants are stringent Etn auxotrophs, as predicted from the scheme shown in Figure 1. The deduced protein sequences of PSD1 and PSD2 showed only 28% identity and 48% similarity in the region of PSD catalytic domain (Figure 2) (18). PSDs from various sources are characterized by an absolutely conserved GST/S motif near the C-terminus of the proenzyme, which is the cleavage site for the autocatalytic formation of the α- and the β-subunits of these enzymes (Figure 2) (42,43). The cleavage reaction results in the concerted production of a covalently attached pyruvoyl prosthetic group that is the catalytic center of the enzyme. In addition, eukaryotic type I PSDs contain mitochondrial targeting sequences and IMM sorting sequences. In contrast, type II PSDs harbor putative targeting sequences for the endomembrane system and are characterized by long N-terminal domains which bear no similarity to type I PSD enzymes. The cleavage motif of type I PSDs is usually composed of the sequence LGST, and in yeast Psd1p this motif is present at amino acids 461–464 (17,41). Type II PSDs typically have GGST as the consensus sequence for internal cleavage, and as shown in Figure 2, yeast Psd2p contains a GGST motif at amino acid residues 1041–1044 (18). Motifs important for substrate binding and catalysis are located in the small α-subunit of the enzyme in close vicinity to the catalytic carbonyl group of the pyruvoyl moiety (42,44). Several functional domains that are not essential for catalytic function have been characterized in Psd2p,
Figure 2: Domain structure and features of Psd2p. Conserved domains and the Pfam domain database designation are highlighted. The C2-1 and C2-2 regions are homologous to those of C2 domains that bind Ca2+ and phospholipid, and also serve as protein–protein interaction domains. The autoendoproteolytic cleavage site, N-terminal to the conserved Ser residue, is denoted with an asterisk. The cleavage at this site occurs in a concerted reaction that generates α- and β-subunits and forms a pyruvoyl prosthetic group on the α-subunit as indicated in the figure. Traffic 2015; 16: 123–134
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and include two conserved C2 domains that are critical for transport of PtdSer to the catalytic domain (37,40). Most characterized C2 domains are Ca2+ -dependent phospholipid-binding modules that participate in protein–phospholipid and protein–protein interactions (45,46). Initially, only one C2 domain (now referred to as C2-2) was identified after elucidation of the deduced sequence of yeast Psd2p (18). With the expansion of structural information about C2 domains, an additional cryptic C2 domain (C2-1) was later identified at the extreme N-terminus of Psd2p (40). Both of these domains were deleted independently and shown to be necessary for in vivo Psd2p function, measured as the ability to rescue the Etn auxotrophy of psd1Δ psd2Δ strain (37,40). However, these C2 domains are dispensable for catalytic activity, when measured in cell extracts, or when assayed in permeabilized cells with the transport-independent substrate NBD-PtdSer, which freely partitions into membranes (37,40).
Localization of Psd2p Initial studies examining the subcellular localization of Psd2p revealed that its distribution overlaps primarily with a marker for vacuoles (>65%) but a significant proportion (35%) also co-fractionated with markers for the Golgi complex, leading to the conclusion that Psd2p is distributed in these compartments (12). Further experiments with Psd2p revealed that it contains a putative Golgi retention sequence (EFDIYNEDEREDSDFQSK) with similarity to a Kex2p sequence (EFDIIDTDSEYDSTLDNK) implicated in its membrane targeting (18,47). Subcellular fractionation experiments demonstrated the colocalization of a subpopulation of Psd2p molecules with Kex2p in the Golgi complex; however, removal of the putative Golgi retention sequence from Psd2p did not alter its localization or function in transport-dependent PtdEtn synthesis (37). Data from Gulshan et al. (13) showed that GFP-tagged Psd2p-expressing cells exhibited punctate fluorescence within the cytoplasm. Further, to determine whether Psd2p-GFP might be localized within the endocytic system, colocalization studies were performed using chimeric endosomal compartment proteins Tlg1- and Tlg2-mCherry. The strains expressing Psd2-GFP formed only a few punctuate structures in the cytoplasm and 126
most of them colocalized with Tlg1 and Tlg2 proteins. Additional studies colocalized Psd2p with a binding protein, Pdr17p/PstB2p (described later) in the endocytic system. These fluorescent microscopic data support the conclusion that both Psd2p and PstB2p are localized to endosomes in addition to elements of the Golgi and vacuolar compartments as suggested previously (48).
psd2𝚫 strains as tools to study ER to mitochondria lipid transport In yeast strains harboring a psd2Δ mutation the only route for decarboxylating PtdSer requires transport of the lipid from the ER to the mitochondria. However, yeast strains can bypass this obligate transport pathway for PtdEtn synthesis, when an external source of Etn is provided. Etn is transported into yeast cells and undergoes further metabolism to phosphoethanolamine (P-Etn) and CDP-Etn and finally PtdEtn by a pathway elucidated by Kennedy and Weiss (21). P-Etn can also be generated by sphingolipid catabolism catalyzed by the dihydrosphingosine-1-phosphate lyase (Dpl1) enzyme (49,50). In general, the Dpl1-based pathway is insufficient to supply enough P-Etn precursor to produce the required pools of PtdEtn needed for cellular growth, when both Psd1p and Psd2p are genetically deleted from cells (18). Supplementation of yeast with low concentrations of lyso-PtdEtn (0.5 mM) can also efficiently rescue the psd1Δ psd2Δ double mutant independently of the Kennedy pathway (38). Lyso-PtdEtn is transported by aminophospholipid flippases at the plasma membrane and acylated by the acyltransferase Ale1p to form PtdEtn (51). The metabolic pathways described above enable selective deletion of either of the PSD genes, producing strains that can be used for mutagenesis and selection of Etn or lyso-PtdEtn auxotrophs with interesting properties. Among the auxotrophic strains, one expects to recover new strains harboring defects in PtdSer transport to Psd1p or Psd2p, and defects in PtdEtn export from the loci of the PSD enzymes to the PtdEtn methyltransferases used to synthesize PtdCho (24). In one application of this genetic screen using a psd2Δ yeast strain, an Etn auxotroph designated pstA1 was recovered (36). The pstA1 strain expressed normal levels of Psd1p, but was defective in converting Traffic 2015; 16: 123–134
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newly synthesized PtdSer to PtdEtn. The gene complementing the growth defect (Etn auxotrophy) of the pstA1 strain is MET30. The complementing protein, Met30p, is an F-box protein subunit of an SCF (Skp1p, Cullin, F-box) ubiquitin ligase (52). Typically, F-box proteins specify target proteins as substrates for the ligase complex (53). In vitro reconstitution studies with purified MAMs and purified mitochondria revealed that the action of Met30p is required for making both donor membranes and acceptor membranes competent for the PtdSer transfer process (36). Thus, this genetic screen in the psd2Δ background has revealed that PtdSer transport to mitochondrial Psd1p is regulated by ubiquitination, although the targets of this ubiquitination have thus far proved elusive. In addition to the forward genetic approaches outlined above, a synthetic biology approach, complemented by high-throughput synthetic genetic studies, has revealed another major determinant of lipid transport between ER and mitochondria. Kornmann et al. (54) generated a yeast strain engineered to express an artificial protein capable of tethering the ER and mitochondria, and searched for mutants in which growth was dependent on the presence of this chimeric linker protein. This led to identification of the ‘ER–mitochondria encounter structure’ or ERMES, which has been proposed to facilitate lipid exchange between these organelles, as outlined in a previous review in this series (55). The ERMES complex consists of Mmm1p, Mdm10p, Mdm12p, Mdm34p and Gem1p (54,56) and is proposed to form a physical bridge between the organelles. Components of the complex were initially annotated as being involved in the maintenance of proper mitochondrial morphology (57–62), because yeast strains defective in any member of this complex display altered mitochondrial morphology, leading to poor growth under respiratory conditions with lactate as sole carbon and energy source (54). Similarly, strains deleted for any ERMES component also showed defects in assembly of the electron transport chain. The identification of ERMES led to the hypothesis that it is involved in phospholipid exchange between the ER and mitochondria. Kornmann et al. (54) studied phospholipid exchange by measuring the rate of PtdSer to PtdCho conversion, which is decreased when ERMES is rendered non-functional. However, other studies of ERMES complex mutants have concluded that PtdSer to PtdEtn conversion rates (both in vivo and in vitro) are not Traffic 2015; 16: 123–134
affected (63,64), leading to the conclusion that alternate, ERMES-independent pathways exist to transport PtdSer between the ER and mitochondria.
psd1𝚫 strains as tools to study lipid transport between the ER and Golgi/endosome In yeast strains with a psd1Δ mutation, the synthesis of PtdEtn from PtdSer requires interorganelle transport of the precursor to the Psd2p enzyme (12,18). When psd1Δ strains are grown in medium devoid of Etn, the PtdSer transport step becomes essential for the synthesis of PtdEtn, and cell growth. In principle, strains with defects in the transport step should be recoverable as Etn (or lyso-PtdEtn) auxotrophs. The application of mutagenesis and screening for obligate Etn auxotrophs, using psd1Δ strains, led to the identification of a strain designated pstB1. The pstB1 strain is defective in the transport-dependent conversion of nascent PtdSer to PtdEtn. The gene complementing the pstB1 defect was identified as STT4, which encodes a PtdIns-4-kinase. The pstB1 strain is defective in PtdIns-4-kinase and the genetic lesion is due to a hypomorphic allele of the STT4 locus (34,65). These findings implicate polyphosphoinositides as regulators of PtdSer transport between the ER and Psd2p, although the mechanism by which this occurs is unclear. Localization studies of Stt4p, conducted with either a fluorescent binding protein or a GFP fusion chimera, identify the plasma membrane as the major subcellular site for this enzyme (66). These localization studies implicate PtdIns-4-kinase generated at the plasma membrane as a regulator of PtdSer transport to Psd2p, but how this occurs remains uncertain. A second mutant yeast strain uncovered by the genetic screen described above was designated pstB2 (35). This strain has wild-type catalytic activity for Psd2p, but is defective in transporting PtdSer to the enzyme, and therefore shows a significant reduction in PtdEtn synthesis from PtdSer. The PSTB2 gene was identified by screening for plasmids capable of rectifying Etn auxotrophy (35). The gene was also identified independently in drug resistance screens, and in assignment of yeast homologs of the SEC14 gene encoding the PtdIns/PtdCho transfer protein (67,68). Similar to Sec14p, PstB2p is a lipid-binding protein that can transfer PtdIns in vitro (35,68). However, PstB2p does not exhibit the PtdCho transfer property of Sec14p. Furthermore, despite being involved in the process of PtdSer 127
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transport, PstB2p does not show high-affinity binding to the lipid, and does not appear to act as a soluble carrier of the lipid.
Reconstitution Studies Examining PtdSer Transport to Psd2p To understand PtdSer transport in more detail, permeabilized cells were used as a starting point for probing the process (69). In contrast to the robust transport of PtdSer to Psd1p in permeabilized cells (69), the activity of PtdSer transport to Psd2p was initially feeble (48). Key elements for successfully restoring transport in permeabilized cells and to developing a useful assay were the inclusion of Mn2+ in the reaction and conduct of the reactions at pH 8.0 (48). With the latter conditions, both the synthesis of PtdSer, and its transport to, and decarboxylation by Psd2p could be followed using a 3 H-serine precursor. Membranes harboring Psd2p (i.e. acceptors) could be removed from permeabilized cells with a simple centrifugation washing step, and subsequently concentrated by high-speed centrifugation. In contrast, membranes necessary for the synthesis of PtdSer (donors) were retained in washed permeabilized cells. The ability to temporally and spatially segregate donor and acceptor compartments for lipid transport also enabled more detailed biochemical and genetic analysis of the process (48). These studies revealed the following important characteristics of the PtdSer transport process: (i) neither ATP nor cytosol is required for transport, (ii) PstB2p association with the acceptor membrane, but not the donor membrane is required, (iii) both C2 domains of Psd2p are required and (iv) Mn2+ is required. Additional reconstitution studies performed with acceptor membranes prepared as described above have successfully made use of artificial donor membranes in the form of PtdSer liposomes (70). Quite surprisingly, a critical feature for liposomes to act as effective donors of PtdSer to membranes harboring Psd2p is a relatively planar geometry (∼400 nm diameter). Liposomes with a high degree of curvature (25–50 nm diameter) are very poor as donors. Similar to studies with permeabilized cells, Mn2+ plays an important role, and its inclusion in the transport reaction doubles the apparent rate of PtdSer transport. An especially curious phenomenon with liposome 128
donors is that dilution of the PtdSer pool within the liposome membrane by other lipids (e.g. PtdCho) disproportionately inhibits transport. For example, donor liposomes consisting of 50% PtdSer and 50% PtdCho fail to transport PtdSer to Psd2p-containing membranes. In contrast to the effects of PtdCho, PtdOH addition to liposomes enhances the transport of PtdSer to acceptor membranes. These data suggest that membrane domains enriched in PtdSer and PtdOH are preferred regions for interaction with acceptor membranes for PtdSer transport. One further characteristic of the liposome donor system is its recapitulation of the genetic requirements of the acceptor membrane for PtdSer transport. Acceptor membranes derived from yeast strains lacking a C2 domain in Psd2p, or lacking PstB2p, are incompetent to transport PtdSer from liposome donors (35,37,48,70). The latter data suggest that PstB2p and the C2 domains of Psd2p constitute a core structure that is essential for PtdSer transport.
Genetic and Physical Interactions of Psd2p and PstB2p As described above, genetic and biochemical studies (34,35,37,48,70) demonstrated the essential nature of PstB2p in the transport of nascent PtdSer from the ER to the locus of Psd2p. As part of a study to define the physical interaction network of proteins that had been implicated in PSTB pathway function, our laboratory performed a two-hybrid screen for PstB2p-interacting proteins (40). Initially, the study identified the conserved but previously uncharacterized open reading frame YPL272C, which we named PBI1 (PstB2 interacting 1). Pbi1p encodes a protein of 517 amino acids and bears a domain common to small molecule N- and O-acetyltransferases (Pfam domain 07247). Other yeast acetyltransferases harboring this domain include Atf1p and Atf2p, which are involved in the formation of volatile alcohol-acetate esters during beer and wine fermentation (71), and Sli1p, which N-acetylates and inactivates the serine palmitoyltransferase inhibitor myriocin (72). It is currently unknown whether Pbi1p is a functional acetyltransferase or if such a putative activity plays a role in lipid transport. However, a previous study examining transcriptional responses to antifungal compounds (73) showed that the PBI1 transcript is induced ∼20-fold upon treatment with the ergosterol biosynthesis Traffic 2015; 16: 123–134
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inhibitor ketoconazole, suggesting that the gene is involved in, or coordinately regulated with sterol biosynthesis, or is involved in the detoxification of ergosterol biosynthetic intermediates accumulating in ketoconazole-treated cells. Pbi1p was produced as a His-tagged soluble protein in Escherichia coli and shown to interact with liposomes containing phosphatidic acid (PtdOH), consistent with a role for PtdOH as a regulator of PSTB pathway function in an in vitro-reconstituted lipid transport system as described above (70). The physical interaction of Pbi1p with PstB2p, the lipid-binding properties of Pbi1p and the apparent lipid-related gene expression changes associated with the PBI1 gene all suggest an unrecognized role for Pbi1p in lipid metabolism. However, deletion of PBI1 did not alter PSTB pathway transport activity as measured in permeabilized cells. In addition, a psd1Δ pbi1Δ double mutant was prototrophic for Etn, demonstrating that the PstB2p–Pbi1p interaction is not essential for lipid transport through the PSTB pathway. Further, two-hybrid analysis was conducted using the mating-based split ubiquitin system (mbSUS) (74) to define interactions between proteins that had been implicated in PSTB pathway function. To this end, a directed mbSUS screen was conducted to define the interaction network between Psd2p, PstB2p, Pbi1p, Stt4p, Scs2p, Osh1p, Osh2p and Osh3p. The latter four proteins had not been demonstrated to have a role in PSTB pathway function, but were included because of their previously annotated physical and/or genetic interactions with Stt4p, whose involvement in the PSTB pathway is supported by genetic studies, but its mechanistic biochemical role remains unclear. This screen confirmed previously described interactions between Psd2p and PstB2p, and unexpectedly identified an interaction between Pbi1p and Scs2p, further suggesting a role for Pbi1p in lipid metabolism. A current model for the formation or maintenance of a membrane contact site between the ER and vesicles containing Psd2p is presented in Figure 3, and includes all known essential, and non-essential, protein and lipid components that have been implicated in this transport process. Perhaps, the most interesting aspect of the physical interactions shown in Figure 3 is that they provide a means for structurally tethering the ER to membranes harboring Psd2p. Two protein constituents of the model are confined Traffic 2015; 16: 123–134
Figure 3: Summary of protein and lipid interactions implicated in PtdSer transport to the locus of Psd2p. PtdSer is synthesized in the ER and Psd2p is localized to the Golgi and endosomal compartments. The N-terminal segment of Psd2p contains two C2 domains (C2-1 and C2-2) that serve as the primary interaction sites with the lipid-binding protein PstB2p. Both Psd2p and PstB2p colocalize to the Golgi and endosomes. The protein Pbi1p is a binding partner to both PstB2p and Scs2p. Scs2p is an integral membrane protein of the ER. These physical interactions can serve to tether the ER with the Golgi and endosomal compartments. Another binding partner for Scs2p is the PtdIns 4-kinase Stt4p, which has been implicated by genetic and biochemical studies as essential for PtdSer transport to the locus of Psd2p. PstB2p, the N-terminal domain of Psd2p, and Pbi1p also bind to PtdOH and these interactions may also serve to tether the ER to the Golgi/endosomes. to the Golgi/endosome, the Psd2p enzyme and its binding partner PstB2p (13,40). Within the Psd2p protein, the C2-1 and C2-2 domains are required for binding to PstB2p. At the ER, Scs2p serves as one sight for coupling this membrane to Psd2p. Data from other studies have identified Scs2p as a binding protein for Stt4p, and this physical interaction may play a role in generating a pool of PtdIns-4-P at the ER that is somehow coupled to PtdSer transport processes. The Pbi1p can bridge the ER to membranes containing Psd2p by its dual interactions with Scs2p and PstB2p. Collectively, the physical interactions shown in Figure 3 integrate both enzymatic and genetic data. However, two of the constituents in Figure 3, Pbi1p and Scs2p, are not essential for PtdSer transport. The latter data suggest that other protein and lipid elements can contribute to the tethering process. 129
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At least three lipids have been implicated in the transport reaction. Although PtdSer is the cargo for transport, physically segregated domains highly enriched in this lipid may also play a role in promoting the process (70). Genetic studies implicate PtdIns 4-P, but the organelle location of this lipid and the mechanism by which it acts, continue to be unclear (34). PtdOH binds to PstB2p, Pbi1p and Psd2p and may function as an alternate mechanism for tethering the ER to membranes containing Psd2p (40).
What Is the Cellular Function of PtdEtn Derived from the PstB/Psd2p Pathway? In considering the relatively complicated nature of de novo, transport-dependent PtdEtn synthesis in yeast described above (Figure 1), a series of related questions arises including: (i) Why do yeast cells maintain multiple, apparently redundant pathways for PtdEtn synthesis? (ii) Are there specific functions of the products of these different pathways? (iii) Are there unique regulatory processes for the biosynthetically distinct pools of PtdEtn? The answers to these questions are unsettled. However, numerous studies of individual enzymes and PtdEtn biosynthetic pathways, complemented by high-throughput genetic and physical interaction studies, have led to the conclusion that each distinct PtdEtn biosynthetic pathway leads to the formation of a PtdEtn pool with specific functions [e.g. supporting mitochondrial function (18,20,22) or vacuolar function (13)], as well as functions that are shared across different biosynthetic pools [ability to serve as a precursor for PtdCho biosynthesis (12)]. To assess the potential role of PtdEtn synthesized by Psd2p, we compiled a list of 75 genes that have been demonstrated in synthetic genetic studies (66–70,75–78) to produce negative growth interactions with psd2Δ mutations. An analysis of this gene set with algorithms from the YeastNet V.3 (http://www.inetbio.org/yeastnet/search.php) is presented in Table 1, and indicates that removal of Psd2p function is generally deleterious when combined with loss-of-function mutations in genes associated with endosomes and retrograde-directed compartments of the Golgi complex. Mutants defective in specific retrograde vesicular transport steps show synergistic growth defects when combined with psd2Δ mutations. This finding is consistent with observations that loss of Psd2p function 130
Table 1: Gene ontology categories (cellular component and biological process) of 75 genes identified as being enriched in negative genetic interactors of psd2Δ in high-throughput synthetic genetic studies GO designation GO:0005783 GO:0005794 GO:0000139 GO:0005789 GO:0034066 GO:0031201 GO:0043529 GO:0006890 GO:0006888 GO:0042147 GO:0048280 GO:0006656 GO:0006986 GO:0016192
GO term: cellular components C:endoplasmic reticulum C:Golgi apparatus C:Golgi membrane C:endoplasmic reticulum membrane C:Ric1p–Rgp1p complex C:SNARE complex C:GET complex GO term: biological process P:retrograde vesicle-mediated transport, Golgi to ER P:ER to Golgi vesicle-mediated transport P:retrograde transport, endosome to Golgi P:vesicle fusion with Golgi apparatus P:phosphatidylcholine biosynthetic process P:response to unfolded protein P:vesicle-mediated transport
p-Value 4.51E−07 5.99E−07 3.59E−06 5.95E−06 0.0001733 0.000292 0.0005155 1.33E−06 8.87E−06 9.51E−05 0.0001195 0.0002512 0.0002512 0.0004824
causes a defect in vacuolar transport functions required for heavy metal detoxification (13), and supports the idea that Psd2p, in complex with PstB2 and potentially other proteins, functions to maintain a lipid environment conducive to proper functioning of the endomembrane system.
Unresolved Questions and Future Directions for Studies on Psd2p As described above, the current model for PtdSer transport to Psd2p is one in which the N-terminal portion of the enzyme acts as a hub for assembly of a multisubunit complex, which facilitates PtdSer transport from the ER to drive PtdEtn synthesis in compartments of the Golgi and endosomes. This Psd2p-specific pool of PtdEtn is likely to be important for maintenance of proper lipid compositions in endosomes and retrograde-directed Golgi vesicles, as suggested by the prevalence of genes specifying these functions as negative genetic interactors of psd2Δ (Table 1). While the studies summarized in this review provide many details regarding PtdSer transport and the cellular function of PtdEtn generated by Psd2p, several Traffic 2015; 16: 123–134
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important questions remain unanswered. The first and most obvious of these questions is: What is the molecular mechanism by which PtdSer is transported? The biochemical and genetic studies outlined above have defined a set of requirements necessary for PtdSer transport to occur; however, the precise mechanism by which PtdSer is transported remains obscure. A second major unresolved question is: How is PtdSer transport and Psd2p catalytic activity regulated? Coordinate regulation of lipid synthesis with other biosynthetic functions is thought to be critical to proper regulation of cell growth and division, and as such, enzymes such as Psd2p are likely to be regulated to meet the demand of the cell for membrane biogenesis. Recent studies examining the regulation of the phospholipid methyltransferase Pem2p/Opi3p identify a role for the oxysterol-binding protein homolog Osh3p as a regulator of this methyltransferase at membrane contact sites between the cortical ER and plasma membrane (73). These observations lead to the conclusion that regulation of phospholipid biosynthesis in yeast and other eukaryotes is driven not only by regulation of the activity of different synthetic enzymes but also through regulation of the transport pathways and membrane contact sites that give those enzymes access to their substrates. Several examples have been outlined in this work, in which we have focused on transport through the Psd2p transport complex, highlighting the necessity for a clearer understanding of interorganelle phospholipid transport in eukaryotic cells, as these processes and the membrane contact sites through which they occur appear to be emerging as important control points for proper membrane biogenesis and function.
Acknowledgments This work was supported by National Institutes of Health Grants 5R37 GM32453 and GM081461 (to D. R. V.) and F32 GM076798 (to W. R. R), and by American Cancer Society Great-West Postdoctoral Fellowship Award PF-06-288-01-CSM (to W. R. R.).
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Review
Transport of Phosphatidylserine from the Endoplasmic Reticulum to the Site of Phosphatidylserine Decarboxylase2 in Yeast Muthukumar Kannan1 , Wayne R. Riekhof2,∗ and Dennis R. Voelker1,∗ 1 Department 2 School ∗
of Medicine and Program in Cell Biology, National Jewish Health, Denver, CO 80206, USA of Biological Sciences, University of Nebraska, Lincoln, NE 68588, USA
Corresponding authors: Dennis R. Voelker, [email protected] and Wayne R. Riekhof, [email protected]
Abstract Over the past two decades, most of the genes specifying lipid synthesis
non-catalytic N-terminal domains act as a hub to nucleate the assembly
and metabolism in yeast have been identified and characterized. Several
of a multiprotein complex, which facilitates PtdSer transport at mem-
of these biosynthetic genes and their encoded enzymes have provided
brane contact sites between the ER and Golgi/endosome membranes.
valuable tools for the genetic and biochemical dissection of interor-
After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to
ganelle lipid transport processes in yeast. One such pathway involves
form PtdEtn, which is disseminated throughout the cell to support the
the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticu-
structural and functional needs of multiple membranes.
lum (ER), and its non-vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine
Keywords endoplasmic reticulum, endosomes, Golgi apparatus, lipid transport, phosphatidylethanolamine, phosphatidylserine decarboxylases
their role in studies of the transport-dependent pathways of de novo syn-
Received 23 September 2014, revised and accepted for publication 28
thesis of phosphatidylethanolamine (PtdEtn). The emerging picture of
October 2014, uncorrected manuscript published online 30 October
the Psd2p-specific transport pathway is one in which the enzyme and its
2014, published online 11 December 2014
Identification of Psd1p, Psd2p and Their Application as Reporters of PtdSer Traffic
as the major sites of glycerophospholipid biosynthesis in eukaryotes (9–11). In yeast, a significant proportion of total PtdEtn can also be synthesized in the compartments of the Golgi and endosomes (12,13). The compartmentation of lipid-synthesizing activities necessitates efficient interorganelle transport to membranes incapable of autonomous lipid synthesis, in order to maintain membrane biogenesis and function.
Phosphatidylethanolamine (PtdEtn) is an abundant membrane phospholipid of yeast as well as many lower and higher eukaryotes and bacteria. In addition to acting as a structural constituent of membranes, PtdEtn is involved in numerous specific cellular functions including autophagy (1,2), fusion and fission events of membranes (3), vacuolar protein delivery (4), the stabilization of membrane proteins or enzymes (5,6) and cell signaling (7,8). The endoplasmic reticulum (ER) and mitochondria are generally accepted
The topological segregation of yeast enzymes involved in aminoglycerophospholipid synthesis is shown in Figure 1. In this scheme, phosphatidylserine synthase (Pss1p, encoded by the CHO1/PSS1 gene) (14) is localized in the www.traffic.dk 123
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exported to other membranes to fulfill their requirements for PtdEtn (23). Many eukaryotes, including yeast, can efficiently methylate PtdEtn to form phosphatidylcholine (PtdCho) in the ER (24).
Figure 1: Topology of de novo PtdCho and PtdEtn biosynthesis in yeast. Major pathways of aminoglycerophospholipid synthesis are depicted, with an emphasis on transport-dependent enzymatic reactions. Interorganelle transport events are denoted with dashed lines, and enzymes are denoted with the standard yeast genetic nomenclature. Pss1p, phosphatidylserine synthase; Psd1p, phosphatidylserine decarboxylase 1; Psd2p, phosphatidylserine decarboxylase2; Pem1p, phosphatidylethanolamine methyltransferase 1; Pem2p, phosphatidylethanolamine methyltransferase 2. The Kennedy pathways constitute reactions for the synthesis of PtdEtn and PtdCho in the ER that proceed through phospho-ethanolamine/choline and CDP-ethanolamine/choline intermediates. ER and mitochondria-associated ER membranes (MAMs) (15,16). Phosphatidylserine decarboxylase1 (Psd1p) is present in mitochondria (17), and phosphatidylserine decarboxylase2 (Psd2p) is distributed in the Golgi and endosomes (12,13,18). Phosphatidylethanolamine methyltransferases 1 and 2 (encoded by the CHO2/PEM1 and OPI3/PEM2 genes) reside in the ER (16), as corroborated by green fluorescent protein (GFP) localization studies (19). Following the synthesis of phosphatidylserine (PtdSer) in the ER, this lipid must be transported to either mitochondria or Golgi/endosome compartments for decarboxylation to generate PtdEtn. The PtdEtn produced within the mitochondria is required for the optimal function of this organelle (17,20), and yeast strains lacking Psd1p undergo conversion to petite phenotypes with high frequency. PtdEtn produced in the ER by the CDP-ethanolamine (Etn)-dependent ‘Kennedy’ pathway (21) is only poorly transported into mitochondria, and cannot fully restore all functions of the pool produced within the mitochondria (12,17,20,22). PtdEtn synthesized by Psd1p within the mitochondria is not static, and can be 124
The Golgi apparatus also has a limited capacity to synthesize some glycerophospholipids (25,26). In yeast, PtdSer transported to the Golgi can also be a substrate for Psd2p (12). The flux of PtdSer through the Psd2p enzyme accounts for up to 30% of the total de novo biosynthetic flux (13), when the Psd1p enzyme is also active, and the resultant PtdEtn can fulfill most cellular needs for this lipid. However, similar to the Kennedy pathway-derived PtdEtn pool, PtdEtn derived from Psd2p is insufficient to completely provide the PtdEtn pool required by mitochondria under respiratory conditions (20,22). When required, the PtdEtn produced by Psd2p in the Golgi/endosome can be transported to the ER for methylation to PtdCho, and serve to fulfill all the cellular needs for these two lipids under non-respiratory conditions (e.g. glucose as carbon source) (12,18).
Biochemical Function of Phosphatidylserine Decarboxylases PSD enzymes play a central role in the phospholipid metabolism of organisms ranging from bacteria to humans, and a similar enzymatic function is likely to be involved in archaeal ether lipid synthesis (27). As described above and in Figure 1, the yeast Saccharomyces cerevisiae expresses two different PSD activities: one associated with the inner mitochondrial membrane (IMM) (Psd1p) (17), as well as a non-mitochondrial enzyme (Psd2p) associated with the Golgi and endosomes (12,13). The PSD enzymes decarboxylate only the lipid-linked form of the serine moiety. Derivatives of PtdSer, including serine, phosphoserine and glycerophosphoserine, fail to serve as either substrates or inhibitors of the catalytic activity (28). Although no extensive surveys of the fatty acid preference of the purified enzymes have been reported, PSDs will utilize substrates containing saturated and unsaturated fatty acids equally well (29). Synthetic substrates such as dimyristoyl PtdSer, 1-acyl2[N(6-[7-nitrobenz-2-oxa-1,3diazo-4-yl]-aminocaproyl)]PtdSer (NBD-PtdSer) (30) and 1-acyl-2-ω-pyreneacyl-PtdSer (31) are also recognized and decarboxylated Traffic 2015; 16: 123–134
Phosphatidylserine Transport to the Locus of Psd2p
by the enzyme, suggesting that the minimum requirement for substrate recognition is a glycerophosphoserine moiety to which two hydrophobic substituents are esterified.
Role of PtdSer Decarboxylases in Transport-Dependent Metabolism of Aminoglycerophospholipids The identification and characterization of genes encoding the yeast PSD enzymes provided important tools for the study of aminoglycerophospholipid transport, facilitating genetic studies, in which mutants could be isolated by simple auxotrophic screens. The genetic manipulation (deletion and/or overexpression) of PSD genes in yeast has thus served as a valuable tool for studying phospholipid transport among different organelles (32,33). The pathways for lipid transport have been conceptually divided into two major branches for simplicity. The ‘A’ pathway describes the movement of PtdSer into- and PtdEtn out of the mitochondria. The psd2Δ strains serve as a tool to study ‘A’ pathways by forcing all biosynthetic flux of PtdSer through the Psd1p-dependent branch. Similarly, the ‘B’ pathways use psd1Δ strains to force the PtdSer movement into and PtdEtn transport out of the Golgi/endosome. These complementary approaches have allowed the identification of strains with defects in both ‘A’ and ‘B’ pathways, and facilitated the cloning and characterization of the corresponding mutant genes and their products (34–40).
Important Elements of PSD Structure The primary structures of yeast PSDs comprise 500 amino acids in Psd1p and 1138 amino acids in Psd2p, corresponding to molecular masses of 56.5 and 130 kDa, respectively
(17,18,41,42). The first complete eukaryotic gene encoding a PSD (PSD1) was cloned and sequenced from yeast (17). Deletion of this gene revealed a second, non-mitochondrial activity which was denoted Psd2p, and the cloning and characterization of the PSD2 locus followed, and demonstrated that psd1Δ psd2Δ double mutants are stringent Etn auxotrophs, as predicted from the scheme shown in Figure 1. The deduced protein sequences of PSD1 and PSD2 showed only 28% identity and 48% similarity in the region of PSD catalytic domain (Figure 2) (18). PSDs from various sources are characterized by an absolutely conserved GST/S motif near the C-terminus of the proenzyme, which is the cleavage site for the autocatalytic formation of the α- and the β-subunits of these enzymes (Figure 2) (42,43). The cleavage reaction results in the concerted production of a covalently attached pyruvoyl prosthetic group that is the catalytic center of the enzyme. In addition, eukaryotic type I PSDs contain mitochondrial targeting sequences and IMM sorting sequences. In contrast, type II PSDs harbor putative targeting sequences for the endomembrane system and are characterized by long N-terminal domains which bear no similarity to type I PSD enzymes. The cleavage motif of type I PSDs is usually composed of the sequence LGST, and in yeast Psd1p this motif is present at amino acids 461–464 (17,41). Type II PSDs typically have GGST as the consensus sequence for internal cleavage, and as shown in Figure 2, yeast Psd2p contains a GGST motif at amino acid residues 1041–1044 (18). Motifs important for substrate binding and catalysis are located in the small α-subunit of the enzyme in close vicinity to the catalytic carbonyl group of the pyruvoyl moiety (42,44). Several functional domains that are not essential for catalytic function have been characterized in Psd2p,
Figure 2: Domain structure and features of Psd2p. Conserved domains and the Pfam domain database designation are highlighted. The C2-1 and C2-2 regions are homologous to those of C2 domains that bind Ca2+ and phospholipid, and also serve as protein–protein interaction domains. The autoendoproteolytic cleavage site, N-terminal to the conserved Ser residue, is denoted with an asterisk. The cleavage at this site occurs in a concerted reaction that generates α- and β-subunits and forms a pyruvoyl prosthetic group on the α-subunit as indicated in the figure. Traffic 2015; 16: 123–134
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and include two conserved C2 domains that are critical for transport of PtdSer to the catalytic domain (37,40). Most characterized C2 domains are Ca2+ -dependent phospholipid-binding modules that participate in protein–phospholipid and protein–protein interactions (45,46). Initially, only one C2 domain (now referred to as C2-2) was identified after elucidation of the deduced sequence of yeast Psd2p (18). With the expansion of structural information about C2 domains, an additional cryptic C2 domain (C2-1) was later identified at the extreme N-terminus of Psd2p (40). Both of these domains were deleted independently and shown to be necessary for in vivo Psd2p function, measured as the ability to rescue the Etn auxotrophy of psd1Δ psd2Δ strain (37,40). However, these C2 domains are dispensable for catalytic activity, when measured in cell extracts, or when assayed in permeabilized cells with the transport-independent substrate NBD-PtdSer, which freely partitions into membranes (37,40).
Localization of Psd2p Initial studies examining the subcellular localization of Psd2p revealed that its distribution overlaps primarily with a marker for vacuoles (>65%) but a significant proportion (35%) also co-fractionated with markers for the Golgi complex, leading to the conclusion that Psd2p is distributed in these compartments (12). Further experiments with Psd2p revealed that it contains a putative Golgi retention sequence (EFDIYNEDEREDSDFQSK) with similarity to a Kex2p sequence (EFDIIDTDSEYDSTLDNK) implicated in its membrane targeting (18,47). Subcellular fractionation experiments demonstrated the colocalization of a subpopulation of Psd2p molecules with Kex2p in the Golgi complex; however, removal of the putative Golgi retention sequence from Psd2p did not alter its localization or function in transport-dependent PtdEtn synthesis (37). Data from Gulshan et al. (13) showed that GFP-tagged Psd2p-expressing cells exhibited punctate fluorescence within the cytoplasm. Further, to determine whether Psd2p-GFP might be localized within the endocytic system, colocalization studies were performed using chimeric endosomal compartment proteins Tlg1- and Tlg2-mCherry. The strains expressing Psd2-GFP formed only a few punctuate structures in the cytoplasm and 126
most of them colocalized with Tlg1 and Tlg2 proteins. Additional studies colocalized Psd2p with a binding protein, Pdr17p/PstB2p (described later) in the endocytic system. These fluorescent microscopic data support the conclusion that both Psd2p and PstB2p are localized to endosomes in addition to elements of the Golgi and vacuolar compartments as suggested previously (48).
psd2𝚫 strains as tools to study ER to mitochondria lipid transport In yeast strains harboring a psd2Δ mutation the only route for decarboxylating PtdSer requires transport of the lipid from the ER to the mitochondria. However, yeast strains can bypass this obligate transport pathway for PtdEtn synthesis, when an external source of Etn is provided. Etn is transported into yeast cells and undergoes further metabolism to phosphoethanolamine (P-Etn) and CDP-Etn and finally PtdEtn by a pathway elucidated by Kennedy and Weiss (21). P-Etn can also be generated by sphingolipid catabolism catalyzed by the dihydrosphingosine-1-phosphate lyase (Dpl1) enzyme (49,50). In general, the Dpl1-based pathway is insufficient to supply enough P-Etn precursor to produce the required pools of PtdEtn needed for cellular growth, when both Psd1p and Psd2p are genetically deleted from cells (18). Supplementation of yeast with low concentrations of lyso-PtdEtn (0.5 mM) can also efficiently rescue the psd1Δ psd2Δ double mutant independently of the Kennedy pathway (38). Lyso-PtdEtn is transported by aminophospholipid flippases at the plasma membrane and acylated by the acyltransferase Ale1p to form PtdEtn (51). The metabolic pathways described above enable selective deletion of either of the PSD genes, producing strains that can be used for mutagenesis and selection of Etn or lyso-PtdEtn auxotrophs with interesting properties. Among the auxotrophic strains, one expects to recover new strains harboring defects in PtdSer transport to Psd1p or Psd2p, and defects in PtdEtn export from the loci of the PSD enzymes to the PtdEtn methyltransferases used to synthesize PtdCho (24). In one application of this genetic screen using a psd2Δ yeast strain, an Etn auxotroph designated pstA1 was recovered (36). The pstA1 strain expressed normal levels of Psd1p, but was defective in converting Traffic 2015; 16: 123–134
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newly synthesized PtdSer to PtdEtn. The gene complementing the growth defect (Etn auxotrophy) of the pstA1 strain is MET30. The complementing protein, Met30p, is an F-box protein subunit of an SCF (Skp1p, Cullin, F-box) ubiquitin ligase (52). Typically, F-box proteins specify target proteins as substrates for the ligase complex (53). In vitro reconstitution studies with purified MAMs and purified mitochondria revealed that the action of Met30p is required for making both donor membranes and acceptor membranes competent for the PtdSer transfer process (36). Thus, this genetic screen in the psd2Δ background has revealed that PtdSer transport to mitochondrial Psd1p is regulated by ubiquitination, although the targets of this ubiquitination have thus far proved elusive. In addition to the forward genetic approaches outlined above, a synthetic biology approach, complemented by high-throughput synthetic genetic studies, has revealed another major determinant of lipid transport between ER and mitochondria. Kornmann et al. (54) generated a yeast strain engineered to express an artificial protein capable of tethering the ER and mitochondria, and searched for mutants in which growth was dependent on the presence of this chimeric linker protein. This led to identification of the ‘ER–mitochondria encounter structure’ or ERMES, which has been proposed to facilitate lipid exchange between these organelles, as outlined in a previous review in this series (55). The ERMES complex consists of Mmm1p, Mdm10p, Mdm12p, Mdm34p and Gem1p (54,56) and is proposed to form a physical bridge between the organelles. Components of the complex were initially annotated as being involved in the maintenance of proper mitochondrial morphology (57–62), because yeast strains defective in any member of this complex display altered mitochondrial morphology, leading to poor growth under respiratory conditions with lactate as sole carbon and energy source (54). Similarly, strains deleted for any ERMES component also showed defects in assembly of the electron transport chain. The identification of ERMES led to the hypothesis that it is involved in phospholipid exchange between the ER and mitochondria. Kornmann et al. (54) studied phospholipid exchange by measuring the rate of PtdSer to PtdCho conversion, which is decreased when ERMES is rendered non-functional. However, other studies of ERMES complex mutants have concluded that PtdSer to PtdEtn conversion rates (both in vivo and in vitro) are not Traffic 2015; 16: 123–134
affected (63,64), leading to the conclusion that alternate, ERMES-independent pathways exist to transport PtdSer between the ER and mitochondria.
psd1𝚫 strains as tools to study lipid transport between the ER and Golgi/endosome In yeast strains with a psd1Δ mutation, the synthesis of PtdEtn from PtdSer requires interorganelle transport of the precursor to the Psd2p enzyme (12,18). When psd1Δ strains are grown in medium devoid of Etn, the PtdSer transport step becomes essential for the synthesis of PtdEtn, and cell growth. In principle, strains with defects in the transport step should be recoverable as Etn (or lyso-PtdEtn) auxotrophs. The application of mutagenesis and screening for obligate Etn auxotrophs, using psd1Δ strains, led to the identification of a strain designated pstB1. The pstB1 strain is defective in the transport-dependent conversion of nascent PtdSer to PtdEtn. The gene complementing the pstB1 defect was identified as STT4, which encodes a PtdIns-4-kinase. The pstB1 strain is defective in PtdIns-4-kinase and the genetic lesion is due to a hypomorphic allele of the STT4 locus (34,65). These findings implicate polyphosphoinositides as regulators of PtdSer transport between the ER and Psd2p, although the mechanism by which this occurs is unclear. Localization studies of Stt4p, conducted with either a fluorescent binding protein or a GFP fusion chimera, identify the plasma membrane as the major subcellular site for this enzyme (66). These localization studies implicate PtdIns-4-kinase generated at the plasma membrane as a regulator of PtdSer transport to Psd2p, but how this occurs remains uncertain. A second mutant yeast strain uncovered by the genetic screen described above was designated pstB2 (35). This strain has wild-type catalytic activity for Psd2p, but is defective in transporting PtdSer to the enzyme, and therefore shows a significant reduction in PtdEtn synthesis from PtdSer. The PSTB2 gene was identified by screening for plasmids capable of rectifying Etn auxotrophy (35). The gene was also identified independently in drug resistance screens, and in assignment of yeast homologs of the SEC14 gene encoding the PtdIns/PtdCho transfer protein (67,68). Similar to Sec14p, PstB2p is a lipid-binding protein that can transfer PtdIns in vitro (35,68). However, PstB2p does not exhibit the PtdCho transfer property of Sec14p. Furthermore, despite being involved in the process of PtdSer 127
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transport, PstB2p does not show high-affinity binding to the lipid, and does not appear to act as a soluble carrier of the lipid.
Reconstitution Studies Examining PtdSer Transport to Psd2p To understand PtdSer transport in more detail, permeabilized cells were used as a starting point for probing the process (69). In contrast to the robust transport of PtdSer to Psd1p in permeabilized cells (69), the activity of PtdSer transport to Psd2p was initially feeble (48). Key elements for successfully restoring transport in permeabilized cells and to developing a useful assay were the inclusion of Mn2+ in the reaction and conduct of the reactions at pH 8.0 (48). With the latter conditions, both the synthesis of PtdSer, and its transport to, and decarboxylation by Psd2p could be followed using a 3 H-serine precursor. Membranes harboring Psd2p (i.e. acceptors) could be removed from permeabilized cells with a simple centrifugation washing step, and subsequently concentrated by high-speed centrifugation. In contrast, membranes necessary for the synthesis of PtdSer (donors) were retained in washed permeabilized cells. The ability to temporally and spatially segregate donor and acceptor compartments for lipid transport also enabled more detailed biochemical and genetic analysis of the process (48). These studies revealed the following important characteristics of the PtdSer transport process: (i) neither ATP nor cytosol is required for transport, (ii) PstB2p association with the acceptor membrane, but not the donor membrane is required, (iii) both C2 domains of Psd2p are required and (iv) Mn2+ is required. Additional reconstitution studies performed with acceptor membranes prepared as described above have successfully made use of artificial donor membranes in the form of PtdSer liposomes (70). Quite surprisingly, a critical feature for liposomes to act as effective donors of PtdSer to membranes harboring Psd2p is a relatively planar geometry (∼400 nm diameter). Liposomes with a high degree of curvature (25–50 nm diameter) are very poor as donors. Similar to studies with permeabilized cells, Mn2+ plays an important role, and its inclusion in the transport reaction doubles the apparent rate of PtdSer transport. An especially curious phenomenon with liposome 128
donors is that dilution of the PtdSer pool within the liposome membrane by other lipids (e.g. PtdCho) disproportionately inhibits transport. For example, donor liposomes consisting of 50% PtdSer and 50% PtdCho fail to transport PtdSer to Psd2p-containing membranes. In contrast to the effects of PtdCho, PtdOH addition to liposomes enhances the transport of PtdSer to acceptor membranes. These data suggest that membrane domains enriched in PtdSer and PtdOH are preferred regions for interaction with acceptor membranes for PtdSer transport. One further characteristic of the liposome donor system is its recapitulation of the genetic requirements of the acceptor membrane for PtdSer transport. Acceptor membranes derived from yeast strains lacking a C2 domain in Psd2p, or lacking PstB2p, are incompetent to transport PtdSer from liposome donors (35,37,48,70). The latter data suggest that PstB2p and the C2 domains of Psd2p constitute a core structure that is essential for PtdSer transport.
Genetic and Physical Interactions of Psd2p and PstB2p As described above, genetic and biochemical studies (34,35,37,48,70) demonstrated the essential nature of PstB2p in the transport of nascent PtdSer from the ER to the locus of Psd2p. As part of a study to define the physical interaction network of proteins that had been implicated in PSTB pathway function, our laboratory performed a two-hybrid screen for PstB2p-interacting proteins (40). Initially, the study identified the conserved but previously uncharacterized open reading frame YPL272C, which we named PBI1 (PstB2 interacting 1). Pbi1p encodes a protein of 517 amino acids and bears a domain common to small molecule N- and O-acetyltransferases (Pfam domain 07247). Other yeast acetyltransferases harboring this domain include Atf1p and Atf2p, which are involved in the formation of volatile alcohol-acetate esters during beer and wine fermentation (71), and Sli1p, which N-acetylates and inactivates the serine palmitoyltransferase inhibitor myriocin (72). It is currently unknown whether Pbi1p is a functional acetyltransferase or if such a putative activity plays a role in lipid transport. However, a previous study examining transcriptional responses to antifungal compounds (73) showed that the PBI1 transcript is induced ∼20-fold upon treatment with the ergosterol biosynthesis Traffic 2015; 16: 123–134
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inhibitor ketoconazole, suggesting that the gene is involved in, or coordinately regulated with sterol biosynthesis, or is involved in the detoxification of ergosterol biosynthetic intermediates accumulating in ketoconazole-treated cells. Pbi1p was produced as a His-tagged soluble protein in Escherichia coli and shown to interact with liposomes containing phosphatidic acid (PtdOH), consistent with a role for PtdOH as a regulator of PSTB pathway function in an in vitro-reconstituted lipid transport system as described above (70). The physical interaction of Pbi1p with PstB2p, the lipid-binding properties of Pbi1p and the apparent lipid-related gene expression changes associated with the PBI1 gene all suggest an unrecognized role for Pbi1p in lipid metabolism. However, deletion of PBI1 did not alter PSTB pathway transport activity as measured in permeabilized cells. In addition, a psd1Δ pbi1Δ double mutant was prototrophic for Etn, demonstrating that the PstB2p–Pbi1p interaction is not essential for lipid transport through the PSTB pathway. Further, two-hybrid analysis was conducted using the mating-based split ubiquitin system (mbSUS) (74) to define interactions between proteins that had been implicated in PSTB pathway function. To this end, a directed mbSUS screen was conducted to define the interaction network between Psd2p, PstB2p, Pbi1p, Stt4p, Scs2p, Osh1p, Osh2p and Osh3p. The latter four proteins had not been demonstrated to have a role in PSTB pathway function, but were included because of their previously annotated physical and/or genetic interactions with Stt4p, whose involvement in the PSTB pathway is supported by genetic studies, but its mechanistic biochemical role remains unclear. This screen confirmed previously described interactions between Psd2p and PstB2p, and unexpectedly identified an interaction between Pbi1p and Scs2p, further suggesting a role for Pbi1p in lipid metabolism. A current model for the formation or maintenance of a membrane contact site between the ER and vesicles containing Psd2p is presented in Figure 3, and includes all known essential, and non-essential, protein and lipid components that have been implicated in this transport process. Perhaps, the most interesting aspect of the physical interactions shown in Figure 3 is that they provide a means for structurally tethering the ER to membranes harboring Psd2p. Two protein constituents of the model are confined Traffic 2015; 16: 123–134
Figure 3: Summary of protein and lipid interactions implicated in PtdSer transport to the locus of Psd2p. PtdSer is synthesized in the ER and Psd2p is localized to the Golgi and endosomal compartments. The N-terminal segment of Psd2p contains two C2 domains (C2-1 and C2-2) that serve as the primary interaction sites with the lipid-binding protein PstB2p. Both Psd2p and PstB2p colocalize to the Golgi and endosomes. The protein Pbi1p is a binding partner to both PstB2p and Scs2p. Scs2p is an integral membrane protein of the ER. These physical interactions can serve to tether the ER with the Golgi and endosomal compartments. Another binding partner for Scs2p is the PtdIns 4-kinase Stt4p, which has been implicated by genetic and biochemical studies as essential for PtdSer transport to the locus of Psd2p. PstB2p, the N-terminal domain of Psd2p, and Pbi1p also bind to PtdOH and these interactions may also serve to tether the ER to the Golgi/endosomes. to the Golgi/endosome, the Psd2p enzyme and its binding partner PstB2p (13,40). Within the Psd2p protein, the C2-1 and C2-2 domains are required for binding to PstB2p. At the ER, Scs2p serves as one sight for coupling this membrane to Psd2p. Data from other studies have identified Scs2p as a binding protein for Stt4p, and this physical interaction may play a role in generating a pool of PtdIns-4-P at the ER that is somehow coupled to PtdSer transport processes. The Pbi1p can bridge the ER to membranes containing Psd2p by its dual interactions with Scs2p and PstB2p. Collectively, the physical interactions shown in Figure 3 integrate both enzymatic and genetic data. However, two of the constituents in Figure 3, Pbi1p and Scs2p, are not essential for PtdSer transport. The latter data suggest that other protein and lipid elements can contribute to the tethering process. 129
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At least three lipids have been implicated in the transport reaction. Although PtdSer is the cargo for transport, physically segregated domains highly enriched in this lipid may also play a role in promoting the process (70). Genetic studies implicate PtdIns 4-P, but the organelle location of this lipid and the mechanism by which it acts, continue to be unclear (34). PtdOH binds to PstB2p, Pbi1p and Psd2p and may function as an alternate mechanism for tethering the ER to membranes containing Psd2p (40).
What Is the Cellular Function of PtdEtn Derived from the PstB/Psd2p Pathway? In considering the relatively complicated nature of de novo, transport-dependent PtdEtn synthesis in yeast described above (Figure 1), a series of related questions arises including: (i) Why do yeast cells maintain multiple, apparently redundant pathways for PtdEtn synthesis? (ii) Are there specific functions of the products of these different pathways? (iii) Are there unique regulatory processes for the biosynthetically distinct pools of PtdEtn? The answers to these questions are unsettled. However, numerous studies of individual enzymes and PtdEtn biosynthetic pathways, complemented by high-throughput genetic and physical interaction studies, have led to the conclusion that each distinct PtdEtn biosynthetic pathway leads to the formation of a PtdEtn pool with specific functions [e.g. supporting mitochondrial function (18,20,22) or vacuolar function (13)], as well as functions that are shared across different biosynthetic pools [ability to serve as a precursor for PtdCho biosynthesis (12)]. To assess the potential role of PtdEtn synthesized by Psd2p, we compiled a list of 75 genes that have been demonstrated in synthetic genetic studies (66–70,75–78) to produce negative growth interactions with psd2Δ mutations. An analysis of this gene set with algorithms from the YeastNet V.3 (http://www.inetbio.org/yeastnet/search.php) is presented in Table 1, and indicates that removal of Psd2p function is generally deleterious when combined with loss-of-function mutations in genes associated with endosomes and retrograde-directed compartments of the Golgi complex. Mutants defective in specific retrograde vesicular transport steps show synergistic growth defects when combined with psd2Δ mutations. This finding is consistent with observations that loss of Psd2p function 130
Table 1: Gene ontology categories (cellular component and biological process) of 75 genes identified as being enriched in negative genetic interactors of psd2Δ in high-throughput synthetic genetic studies GO designation GO:0005783 GO:0005794 GO:0000139 GO:0005789 GO:0034066 GO:0031201 GO:0043529 GO:0006890 GO:0006888 GO:0042147 GO:0048280 GO:0006656 GO:0006986 GO:0016192
GO term: cellular components C:endoplasmic reticulum C:Golgi apparatus C:Golgi membrane C:endoplasmic reticulum membrane C:Ric1p–Rgp1p complex C:SNARE complex C:GET complex GO term: biological process P:retrograde vesicle-mediated transport, Golgi to ER P:ER to Golgi vesicle-mediated transport P:retrograde transport, endosome to Golgi P:vesicle fusion with Golgi apparatus P:phosphatidylcholine biosynthetic process P:response to unfolded protein P:vesicle-mediated transport
p-Value 4.51E−07 5.99E−07 3.59E−06 5.95E−06 0.0001733 0.000292 0.0005155 1.33E−06 8.87E−06 9.51E−05 0.0001195 0.0002512 0.0002512 0.0004824
causes a defect in vacuolar transport functions required for heavy metal detoxification (13), and supports the idea that Psd2p, in complex with PstB2 and potentially other proteins, functions to maintain a lipid environment conducive to proper functioning of the endomembrane system.
Unresolved Questions and Future Directions for Studies on Psd2p As described above, the current model for PtdSer transport to Psd2p is one in which the N-terminal portion of the enzyme acts as a hub for assembly of a multisubunit complex, which facilitates PtdSer transport from the ER to drive PtdEtn synthesis in compartments of the Golgi and endosomes. This Psd2p-specific pool of PtdEtn is likely to be important for maintenance of proper lipid compositions in endosomes and retrograde-directed Golgi vesicles, as suggested by the prevalence of genes specifying these functions as negative genetic interactors of psd2Δ (Table 1). While the studies summarized in this review provide many details regarding PtdSer transport and the cellular function of PtdEtn generated by Psd2p, several Traffic 2015; 16: 123–134
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important questions remain unanswered. The first and most obvious of these questions is: What is the molecular mechanism by which PtdSer is transported? The biochemical and genetic studies outlined above have defined a set of requirements necessary for PtdSer transport to occur; however, the precise mechanism by which PtdSer is transported remains obscure. A second major unresolved question is: How is PtdSer transport and Psd2p catalytic activity regulated? Coordinate regulation of lipid synthesis with other biosynthetic functions is thought to be critical to proper regulation of cell growth and division, and as such, enzymes such as Psd2p are likely to be regulated to meet the demand of the cell for membrane biogenesis. Recent studies examining the regulation of the phospholipid methyltransferase Pem2p/Opi3p identify a role for the oxysterol-binding protein homolog Osh3p as a regulator of this methyltransferase at membrane contact sites between the cortical ER and plasma membrane (73). These observations lead to the conclusion that regulation of phospholipid biosynthesis in yeast and other eukaryotes is driven not only by regulation of the activity of different synthetic enzymes but also through regulation of the transport pathways and membrane contact sites that give those enzymes access to their substrates. Several examples have been outlined in this work, in which we have focused on transport through the Psd2p transport complex, highlighting the necessity for a clearer understanding of interorganelle phospholipid transport in eukaryotic cells, as these processes and the membrane contact sites through which they occur appear to be emerging as important control points for proper membrane biogenesis and function.
Acknowledgments This work was supported by National Institutes of Health Grants 5R37 GM32453 and GM081461 (to D. R. V.) and F32 GM076798 (to W. R. R), and by American Cancer Society Great-West Postdoctoral Fellowship Award PF-06-288-01-CSM (to W. R. R.).
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