Bridging gaps in phospholipid transport

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    the lipids and proteins that must be present onmembranes for inter-organelle phospholipid transport

    Review TRENDS in Biochemical Sciences Vol.30 No.7 July 2005elles in vesicles whose structure is primarily defined byidentifying some of the molecules involved in transportand experimenting to test their mechanisms-of-action.These endeavors are providing a framework for how a fewof these polar lipids can be transported.

    One feature of intermembrane phospholipid transportthat is not widely appreciated is the resistance of theprocess to inhibitors that affect membrane protein andsecretory protein transport. This resistance is surprisingfrom several perspectives. It is well established that manymembrane and secreted proteins travel between organ-

    now implicate specific genes and their products in theprocesses [1721]. Most importantly, the genetic advancesprovide crucially important raw materials for reconstitu-tion studies that can be used to probe the mechanisms ofphospholipid transport.

    Organelle specific metabolism ofaminoglycerophospholipidsOne combined biochemical and genetic approach forexamining phospholipid transport makes use of thelipid transport. This discipline is in the early stages of

    to begin to unravel some of the complexities of phospho-

    continues to be made and a growing number of mutantshave been isolated from mammalian cells and yeast thatto occur between the endoplasmic reticulum andmitochondria or Golgi. These data suggest that proteinand lipid assemblies on donors and acceptors promotemembrane docking and facilitate lipid movement.

    IntroductionThe maturation of organelles within eukaryotic cellsprimarily requires the selective transport of specificproteins and lipids to the limiting membrane and interiorof the developing structures. In the past two decades,large amounts of information and fine mechanistic detailabout protein sorting to many organelles has beenobtained [1,2]. By contrast, our understanding about theprocess of phospholipid transport for the purpose of neworganelle assembly remains small. However, recentadvances, especially the identification of mutant strainsof yeast and mammalian cells, are now providing the toolsBridging gaps in phtransportDennis R. Voelker

    Program in Cell Biology, Department of Medicine, National JewDenver, CO 80206, USA

    Phospholipid transport between membranes is a funda-mental aspect of organelle biogenesis in eukaryotes;however, little is know about this process. A significantbody of data demonstrates that newly synthesizedphospholipids can move betweenmembranes by routesthat are independent of the vesicular traffic that carriesmembrane proteins. Evidence continues to accumulatein support of a system for phospholipid transport thatoccurs at zones of apposition and contact betweendonor membranes the source of specific phospholipids and acceptor membranes that are unable to synthesizethe necessary lipids. Recent findings identify some ofMedical and Research Center, 1400 Jackson St.,

    phospholipid [3]. Thus, some phospholipid movementbetween organelles must occur via vesicles. When theproduction of these vesicles is arrested by mutation [2] orintoxication with poisons such as brefeldin A [4,5], neitherthe lipid in the vesicle nor the membrane proteins orencapsulated cargo proteins are delivered to theirdestinations. However, in several reports the traffic ofbiosynthetically radiolabeled phospholipids betweenorganelles proceeds unabated under the same conditionsthat arrest membrane or secreted protein transport [69].In addition, reconstitution studies with permeabilizedcells and isolated organelles in many cases fail todemonstrate any dependence of newly synthesized phos-pholipid transport upon cytosolic factors, ATP or GTP[1016]. These findings strongly support a mechanism forphospholipid transport between many organelles that canproceed via routes that are independent of vesicleformation, migration and fusion. It is not clear whythere should be a non-vesicular mechanism to movephospholipids between membranes or why it should beso predominant. One possibility is that non-vesicularphospholipid transport might be an evolutionarily primi-tive system for moving components between membranesthat preceded the development of vesicular mechanisms.The efficiency and efficacy of the non-vesicular transportcould be such that it has been retained as a defaultmechanism for the majority of phospholipid transport.

    Progress in addressing the mechanisms of intracellularphospholipid transport in eukaryotes has been hamperedby the lack of strong genetic selections and screens, andalso convenient biochemical methods for measuring theprocesses. Despite these impediments, modest progressspholipidpholipids (Box 1), phosphatidylserine (PtdSer), phospha-tidylethanolamine (PtdEtn) and phosphatidylcholine

    Corresponding author: Voelker, D.R. (voelkerd@njc.org).Available online 13 June 2005

    www.sciencedirect.com 0968-0004/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2005.05.008organelle specific metabolism of the aminoglycerophos-

  • Box 1. Aminoglycerophospholipids

    In many eukaryotic cells, including those from mammals and the yeastSaccharomyces cerevisiae, the aminoglycerophospholipids comprisew7080% of the total phospholipids present in cell membranes. Thestructures of these lipids are shown in Figure I. On average, intracellularmembranes in many eukaryotes have a phospholipid composition of50% PtdCho, 1025% PtdEtn and 110% PtdSer. Yeast can synthesize the

    full complement of their aminoglycerophospholipids by decarboxylat-ing PtdSer and methylating PtdEtn. The methyl groups are transferred toPtdEtn from S-adenosylmethionine. Nucleated mammalian cells alsosynthesizePtdSer and decarboxylate it to formPtdEtn.However,with theexception of hepatocytes, mammalian cells do not synthesize significantamounts of PtdCho from PtdEtn.

    CH

    CH

    H2C

    HC

    H C

    O

    O PO

    O

    R1

    O R 2 CO2NH

    CH

    CH

    H2C

    HC

    H C

    O

    O PO

    O

    R1

    O R 2

    N

    CHCH

    3

    CH

    CH

    H2C

    HC

    H C

    O

    O PO

    O

    R1

    O R 2

    NH

    Phosphatidylserine Phosphatidylethanolamine Phosphatidylcholine

    pha

    tdSer is decarboxylated to form PtdEtn, which is subsequently methylated to form

    eth

    Review TRENDS in Biochemical Sciences Vol.30 No.7 July 2005 397(PtdCho), as shown schematically in Figure 1. In the yeastSaccharomyces cerevisiae, PtdSer is synthesized in theendoplasmic reticulum (ER) and a subdomain of thisorganelle known as the mitochondria-associated mem-brane (MAM) [15]. After its synthesis, PtdSer is trans-ported from the ER and MAM to numerous organellesincluding the plasma membrane, mitochondria and Golgi.Upon arrival of PtdSer at the mitochondria, it is importedto the inner membrane where it becomes a substrate forPtdSer decarboxylase 1 (Psd1p; GenBank accessionnumber: NP_014230) [22], which catalyzes the conversionto PtdEtn. Transport of PtdSer to the Golgi also providesthe substrate for PtdSer decarboxylase 2 (Psd2p;

    2O

    2 3 2O

    Figure I. Phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn) and phos

    pholipids. These lipids are closely related structurally and linked metabolically. P

    PtdCho. The CO2 moiety (red) of PtdSer that is removed to form PtdEtn and the mNP_011686) and generates PtdEtn in this locale [23,24].In either case, the formation of PtdEtn constitutes achemical reporter for the transport of PtdSer to therespective organelles. The subsequent transport of PtdEtnout of the mitochondria or Golgi to the ER results in thefurther metabolism of a significant portion (but not all) of

    PtdSer PtPsd2p

    Ser PtdSerPss1p

    PtdSer PtPsd1p

    Mitochondri

    Golgi

    ER/MAM

    Figure 1. The transport and metabolic itinerary of the aminoglycerophospholipids. PtdSe

    associated membrane (MAM) by the action of PtdSer synthase (Pss1p). After synthesis, th

    is imported to the inner membrane and decarboxylated by the Psd1p to form PtdEtn. Like

    at this location. The PtdEtn is exported from either organelle to the ER, where it is sequ

    PtdCho. The arrows between the organelles define major transport steps for these phosph

    Psd1p and the PtdEtn is subsequently transported out of the mitochondria. In mammal

    www.sciencedirect.comthis lipid to PtdCho by the combined actions of PtdEtnmethyltransferases 1 and 2 (Pem1p and Pem2p;NP_011673 and NP_012607, respectively) [25,26]. Thissynthesis of PtdCho (from what was originally PtdSer)constitutes another chemical reporter for the transport ofPtdEtn from the loci of Psd1p and Psd2p to the ER.Mammalian cells also synthesize PtdSer in a MAMfraction [27] and transport the lipid to the mitochondriawhere it is decarboxylated by Psd1p [28]. However,mammalian cells have not been reported to contain aPsd2p enzyme and, with the exception of hepatocytes, donot methylate the resultant PtdEtn to form PtdCho.Consequently, the majority of studies with mammalian

    yl groups (green) that are added to PtdEtn to form PtdCho are shown.2O

    2 2

    CH3

    3

    2 32

    tidylcholine (PtdCho) comprise a family of lipids known as the aminoglycerophos-cells use the action of Psd1p to examine PtdSer transportto the mitochondria [29].

    Genetic screens for phospholipid transport mutantsThe organelle specific metabolism of PtdSer and PtdEtnhas been used to develop genetic screens in yeast to

    Ti BS

    dEtn

    dEtn

    PtdEtn PtdCho

    Pem1pPem2p

    a

    ER

    r is synthesized in the endoplasmic reticulum (ER) and closely related mitochondria-

    e PtdSer is transported to other organelles. Upon arrival at the mitochondria, PtdSer

    wise, when PtdSer arrives at the Golgi it is decarboxylated by Psd2p to form PtdEtn

    entially methylated in three reactions by PtdEtn methyltansferases 1 and 2 to form

    olipids. All steps shown occur in yeast. In mammalian cells, PtdSer is transported to

    s, only hepatocytes express significant levels of PtdEtn methyltransferase.

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    Review TRENDS in Biochemical Sciences Vol.30 No.7 July 2005398Ser PtdSer PtdSer

    (a) PSTA and PEEA pathways

    Ser PtdSer PtdSer

    (b) PSTB and PEEB pathways

    Mitoc

    MAM

    pstAmutations

    G

    ER

    pstBmutations

    Figure 2. Genetic screening in yeast for phospholipid transport mutants. (a) In psd2D

    PtdSer transport A and PtdEtn export A pathways (PSTA and PEEA), which involve li

    and then screened for Etn auxotrophs. Among the Etn auxotrophs will be new strains

    PSTA portion of the pathway is highly active in mammalian cells and in yeast. (b).identify mutations, genes and proteins that participate inlipid transport. The general features of this screening areoutlined in Figure 2. In yeast that have been manipulatedto delete the PSD1gene encoding Psd1p (i.e. strains with apsd1D mutation), or to delete the PSD2 gene encodingPsd2p (i.e. strains with a psd2D mutation), growth isrelatively normal in minimal media [2224]. By contrast,when doubly mutated psd1D psd2D strains are con-structed, they are unable to synthesize sufficient PtdEtnfor survival in minimal media [23]. However, like manyeukaryotes, yeasts possess multiple pathways for thesynthesis of PtdEtn [30]. One of these pathways, usuallycalled the Kennedy pathway in recognition of the seminalcontributions of Eugene P. Kennedy, enables yeast to useethanolamine (Etn) to synthesize PtdEtn. Empirically, onefinds that the psd1D psd2D double mutant strains of yeastcan be rescued by Etn supplementation [23]. From thesefindings, Trotter and Voelker postulated that if Etn canrescue PtdEtn deficiency due to inactive Psd1p and Psd2pactivity, then it might also rescue PtdEtn deficiency due toa defect in transport of PtdSer to the loci of either Psd1p orPsd2p [18]. From a theoretical standpoint and forsimplicity, the transport of PtdSer to Psd1p was namedthe PtdSer transport A (PSTA) pathway and the transportof PtdSer to Psd2p was named the PtdSer transport B(PSTB) pathway. Likewise, newly formed PtdEtn must beexported from the mitochondria and Golgi back to the ERfor the synthesis of PtdCho and these pathways are namedPEEA and PEEB.

    along the PtdSer transport B and PtdEtn export B pathways (PSTB and PEEB), which in

    mutagenized and then screened for Etn auxotrophs. Among the Etn auxotrophs will be n

    mutants). Abbreviations: ER, endoplasmic reticulum; MAM, mitochondria-associated m

    www.sciencedirect.comTi BS

    PtdEtn PtdEtn

    PtdCho

    Etn

    PtdEtn PtdEtn

    PtdCho

    Etn

    dria ER

    peeAmutations

    i ER

    peeBmutations

    tant strains lacking Psd2p, aminoglycerophospholipid synthesis proceeds along the

    movement into and out of the mitochondria. The psd2D strains can be mutagenized

    h defects in PtdSer transport (pstA mutants) and PtdEtn export (peeA mutants). The

    sd1D mutant strains lacking Psd1p, aminoglycerophospholipid synthesis proceedsIn execution, the screen for yeast mutants defective inPtdSer transport to Psd1p uses strains harboring a psd2Dmutation that are mutagenized and analyzed for Etnauxotrophy [20]. The psd2D genetic background forcesalmost all PtdEtn synthesis to proceed via transport ofPtdSer to Psd1p, as shown in Figure 2a. The mutation ofpsd2D strains and screening for Etn auxotrophs enablesthe identification of new strains defective in the transportof PtdSer to Psd1p, in addition to strains with defects inthe activity of Psd1p. In yeast, this screen has produced amutant named pstA1 (described in more detail later) andits corresponding complementing gene. Similar types ofanalyses can be performed with strains that have a psd1Dmutation, which are dependent on the transport of PtdSerto Psd2p for the synthesis of the majority of their PtdEtn.Mutation and screening for Etn auxotrophs in strains witha psd1Dmutation enables the identification of new strainsthat are defective in transport of PtdSer to Psd2p, or thatare defective in the activity of this enzyme, as illustratedin Figure 2b. Thus far, this second screen has yieldedtwo mutants (pstB1 and pstB2) and their correspondingcomplementing genes, in addition to a variant of Psd2p,that have provided important new insight into the processof PtdSer transport [16,18,19,21]. In principle, both ofthese screens should also yield new mutants (peeA andpeeB) in the export of PtdEtn from the mitochondria andGolgi back to the ER.

    A screen related to that described previously for thePSTA pathway has also been applied to mammalian cells.

    volve lipid movement into and out of the Golgi in yeast. The psd1D strains can be

    ew strains with defects in PtdSer transport (pstB mutants) and PtdEtn export (peeB

    embrane.

  • Review TRENDS in Biochemical Sciences Vol.30 No.7 July 2005 399This screen was designed to identify strains that have aplasma membrane deficiency in PtdEtn and are resistantto the effects of a toxin that binds the lipid. The toxin isthe cyclic peptide Ro090198, which recognizes PtdEtnand causes cytolysis. This screen produced a variant ofCHO-K1 cells (R41) that is defective in PtdSer transport tothe locus of Psd1p and results in a deficiency in cellularPtdEtn [17].

    Biochemical studiesIn addition to the genetic approach, biochemical studieswith intact cells, permeabilized cells and isolated organ-elles have used the action of Psd1p and Psd2p to examineand characterize PtdSer transport. For lipid transport toPsd1p, yeast and mammalian cells exhibit some differ-ences, if the process is dissected spatially and temporally.The biosynthetic machinery for PtdSer is also differentbetween yeast and mammalian cells. Yeast uses a PtdSersynthase (NP_010943), which requires CDP-diacylglycerolas the source of the phosphatidic acid moiety thatcondenses with serine to make PtdSer [30]. This PtdSeris preferentially synthesized in the MAM compartmentof the ER and transferred to the mitochondria in anATP-independent reaction [14,15,31]. Morphological evi-dence indicates that PtdSer transfer occurs at the zones ofapposition between the MAM and the outer mitochondrialmembrane. The machinery that affects the transfer ofPtdSer between the organelles remains unknown butgenetic studies (described later) are providing some cluesabout the regulation of the process. Once the PtdSer isincorporated into the outer mitochondrial membrane,there are no demonstrable energetic requirements for itto gain access to Psd1p present in the inner membrane[11,15]. The mitochondrial outer membrane can also beloaded with a PtdSer analog (1-acyl, 2-(NBD-aminoca-proyl)-PtdSer, and this lipid is also imported to the locus ofPsd1p in an ATP-independent manner [20,32].

    In mammalian systems, studies using metabolicinhibitors with intact cells reveal that there is an ATPrequirement for PtdSer transport to the locus of Psd1p[28]. Evidence for this ATP requirement is also producedin transport studies using permeabilized cells [13,33].However, experiments with liposomal or isolated micro-somal donors and purified mitochondria do not demon-strate an ATP requirement for PtdSer transport betweenthe membranes [12]. These results strongly suggest thatthe terminal event of lipid transfer from a donor mem-brane to the outer mitochondrial membrane does notrequire ATP in either mammalian cells or yeast (asdescribed earlier). However, unlike yeast, mammaliancells require ATP at multiple steps preceding the finallipid transfer between the membranes. Mammalian cellssynthesize PtdSer by an exchange reaction in which theamino alcohol substituents (Etn and Cho) present inPtdEtn and PtdCho are exchanged for Ser [34].

    Thus, in contrast to yeast, the phosphatidic acid moietyof mammalian PtdSer is derived from PtdEtn and PtdCho.The mammalian reaction requires Ca2C and in intact andpermeabilized cells is coupled to the ability of the ER and

    2CMAM to sequester the ion via a Ca -ATPase [13,32].After PtdSer is formed, there is another ATP requirement

    www.sciencedirect.comthat precedes the final lipid transfer to mitochondria. Thisstep is known to be separate from the ATP requirementnecessary for synthesis of PtdSer because it is possible topulse-radiolabel the PtdSer pool and subsequently arrestfurther synthesis of this lipid. Even under conditions ofarrested PtdSer synthesis, the previously synthesizedradioactive lipid is still transported to the mitochondria.The ATP requirement that follows the synthesis of PtdSeris not well understood because isolated mitochondriareadily import PtdSer when incubated with liposomescontaining this lipid in the absence of ATP [12]. Thecurrent assessment of these data is that the newly synthe-sized PtdSer requires ATP to enter a portion of the MAMthat enables it to interact directly with the mitochondria[35]. Although it is tempting to speculate that thenascent PtdSer is lumenally sequestered and requiresthe ATP-dependent action of a transbilayer translocase tomove it to the cytosolic surface of the bilayer, to date, thereis no experimental evidence to support this idea.

    The reconstitution of PtdSer synthesis and transport inpermeabilized mammalian cells has also been used toscreen for macromolecular factors that augment thetransport reaction. A successful application of thisapproach led to the identification in brain cytosol of anEF-hand protein capable of increasing the rate oftransport of PtdSer from the ER and MAM to the mito-chondria [36]. The protein, named S100B (NP_006236),increases the rate of lipid transport approximatelythreefold. It is not known whether this protein actuallyparticipates in the transport reaction or promotes thestability and/or assembly of interactions between the ERor MAM and mitochondria.

    Studies with transport mutantsExamination of the biochemical characteristics of PtdSertransport using the mutants identified in the screensdescribed previously has provided important insights intothe types of molecules involved in the process and theirmechanisms-of-action (Figure 3). The yeast mutant pstA1is an Etn auxotroph that shows a defect in the rate ofconversion of nascent PtdSer to PtdEtn in intact cells [20].The mitochondria purified from the mutant strain have amarkedly reduced level of PtdEtn and examination ofthese mitochondria on sucrose density gradients revealsthat they are much denser than the organelles from wild-type cells. This is expected if the phospholipid:proteinratio is reduced in mutant mitochondria as a consequenceof reduced phospholipid transport to the organelle. PtdSertransport between the outer and inner mitochondrialmembranes is normal in the pstA1 mutant whenmeasured using the analog 1-acyl, 2-(NBD-aminoca-proyl)-PtdSer. Reconstitution experiments using MAMand mitochondria reveal a defect in the transport ofPtdSer between the organelles. When MAM and mito-chondria are purified from wild-type and mutant cells andrecombined in different combinations as donors andacceptors, the lesion in pstA1 maps to both of themembranes. This finding means that the MAM fromthe pstA1 strain are defective as donor membranes, and

    the mitochondria from the pstA1 strain are defective asacceptor membranes. The gene that rectifies all the

  • Review TRENDS in Biochemical Sciences Vol.30 No.7 July 2005400Ser PtdSer

    PtdSer

    ATP

    Met30p

    PtdSer PtdEtnPtdSer

    X Y

    R41p

    PtdIns

    S100B

    Psd1p

    Mitochondria

    Golgi

    MAMphospholipid synthesis and transport abnormalities of thepstA1 mutation was cloned by complementation andidentified as MET30 (NP_012218). MET30 encodes aprotein subunit (Met30p) of a multi-component E3ubiquitin ligase [37]. The Met30p is an F box proteinthat dictates substrate specificity of the ubiquitin ligase[38]. This result is quite striking and provides unantici-pated linkage between protein ubiquitination and phos-pholipid transport. A growing body of data now implicatesprotein ubiquitination in many membrane traffickingevents, including viral budding at the cell surface,endosomal protein sorting and multivesicular bodyformation [39]. Ubiquitination is also known to regulategene expression by controlling the half life and/or activity

    Ti BS

    SerPtdSer

    PtdIns4P

    PtdOH

    PtdEtn

    Stt4p

    Psd2p

    PstB2pPtdSerdomain

    C2domain

    ER

    Adapterproteins?

    Figure 3.Molecular components and properties of PtdSer transport to mitochondria

    and the Golgi. PtdSer synthesized in the mitochondria-associated membrane

    (MAM) domain of the endoplasmic reticulum (ER) is transported to the outer

    mitochondrial membrane under the regulation of yeast Met30p, a ubiquitin ligase

    subunit. Met30p affects the properties of both the MAM and the mitochondria and

    this is proposed to occur via protein components (designated X and Y) on the

    respective organelles. It is not yet known if Met30p ubiquitinates X and Y directly on

    the MAM and mitochondria, or if its action regulates their degradation or

    transcription. The rate of transfer of PtdSer to mammalian mitochondria can be

    augmented several-fold by the action of mammalian S100B, an EF-hand-Ca2C

    binding protein. Genetic screening identifies a CHO-K1 mutant (R41) that is

    proposed to have a lesion in a putative protein, R41p, required for efficient transport

    of PtdSer between the outer (om) and inner (im) mitochondrial membranes. In

    mammalian cells, but not yeast, there is an ATP requirement for newly synthesized

    PtdSer to become competent for transport to the mitochondria. In yeast, PtdSer

    synthesized in the ER is transported to the Psd2p in the Golgi for the synthesis of

    PtdEtn. The Psd2p has not been identified in mammalian cells. Reconstitution

    studies with chemically defined donors demonstrate that PtdSer-rich domains are

    the preferred source of the lipid destined for transport to Psd2p, and PtdIns4P and

    PtdOH can augment the transport reaction. Genetic studies implicate Stt4p as a

    source of Ptd-Ins-4-P required for PtdSer transport but there is uncertainty how this

    occurs because the enzyme is primarily found in the plasma membrane. The

    presence of Psd2p and PstB2p on the Golgi membrane is required for

    intermembrane PtdSer transport to occur. The C2 domain of Psd2p is not required

    for catalysis by the enzyme but is necessary for the transport reaction. The C2

    domain might function by recognizing anionic lipids in the donor membrane and

    promoting docking with the acceptor membranes. It is also likely that adaptor

    proteins have a role in promoting interactions among proteins and lipids between

    the membranes.

    www.sciencedirect.comof transcription factors. One known substrate for E3ubiquitin ligase containing Met30p is the transcriptionfactor Met4p [4042] (NP_014296). Upon ubiquitination,Met4p is inactivated. Biochemical studies reveal that thepstA1 mutant is defective in ubiquitination of Met4p [20].Currently, it is not known how many proteins can serve assubstrates for recognition by Met30p.

    At this stage, it is unclear howMet30p regulates PtdSertransport between the MAM and the mitochondria. Threepossible explanations seem most likely. The first hypoth-esis and perhaps the simplest is that protein substratespresent on both the MAM and mitochondria are recog-nized by Met30p. Ubiquitination of these putative targetproteins (designated as X and Y in Figure 3) could have arole in either direct activation of transport molecules, orMAM-mitochondria recognition and docking for thepurpose of lipid transport. A second hypothesis is thatan inhibitor of PtdSer transport resides on both the MAMand mitochondria. In this case, Met30p directed ubiqui-tination of a protein substrate could lead to its degradationand alleviation of an inhibitory block upon transport. Athird hypothesis is that the action of Met30p upon Met4pregulates transcription of a factor involved in lipidtransport. Because ubiquitination directed by Met30pinactivates Met4p, it is likely that the factor would be anegative regulator of PtdSer transport.

    Biochemical studies in mammalian cells, with the R41mutant cell-line that is resistant to the cytolytic effects ofthe toxin Ro090198, also reveal important informationabout the process of PtdSer transport within the mito-chondria [17]. The R41 cells show a reduction in totalcellular PtdEtn content and the metabolism of nascentPtdSer to PtdEtn. Enzymatic studies reveal that Psd1pactivity is normal but the transport of 1-acyl, 2-(NBD-aminocaproyl)-PtdSer between the outer and innermitochondrial membranes is markedly reduced. Interest-ingly, this defect in lipid import into the mitochondria doesnot affect protein import into the organelle. These resultsprovide significant evidence for the existence of specificproteins involved in the transport of phospholipidsbetween the outer and inner mitochondrial membranesthat are independent of the protein transport machinery.Thus far, the cDNA and gene required to complement theR41 strain have not been cloned.

    Examination of the mutants obtained in the PSTBpathway is also providing some new definition to themolecular requirements for the transport of PtdSer toPsd2p in yeast cells. The first mutant described in thispathway was pstB1 [18]. The pstB1mutant shows a defectin the conversion of nascent PtdSer to PtdEtn by Psd2p.The reduced PtdEtn synthesis is not due to an alterationin Psd2p activity but in the access of the substrate to theenzyme. The gene that complements the pstB1 growthdefect (Etn auxotrophy) and abnormality in lipid syn-thesis is STT4 (NP_013408), which encodes a phospha-tidylinositol 4-kinase (PtdIns4-kinase; Stt4p) [43]. TheStt4p is one of three PtdIns4-kinases that have beendescribed in yeast and shows both plasma membraneand microsomal distribution in subcellular fractions.

    The enzyme is tethered to the plasma membrane bya structural protein named Sfk1p [44] (NP_012873).

  • Review TRENDS in Biochemical Sciences Vol.30 No.7 July 2005 401There is reasonable evidence that the major pool ofphosphatidylinositol 4-phosphate (PtdIns4P) producedby Stt4p resides in the plasma membrane [45]. However,it remains unclear whether the PtdIns4P generated bythis enzyme can also be found in other intracellularmembranes. Although the genetic experiments with pstB1strains implicate PtdIns4P at some stage of PtdSertransport to Psd2p, much remains uncertain. It isunknown whether PtdIns4P or its downstream product,phosphatidylinositol (4,5)-bisphosphate PtdIns(4,5)P2, isimportant in the transport process. Analysis of thesequence of Psd2p reveals the presence of a C2 domain.Typically, C2 domains are involved in Ca2C binding,proteinlipid and proteinprotein interactions [46].Anionic lipids, including PtdSer and polyphosphoinosi-tides, are recognized by C2 domains and this raises thepossibility that Psd2p might participate directly inrecognition of these lipids on the donor membrane aspart of the transport reaction.

    To test the role of the C2 domain of Psd2p in PtdSertransport, deletion mutants lacking this domain(psd2-C2D) were constructed [21]. In strains lackingwild-type copies of Psd1p and Psd2p, the mutant protein,Psd2-C2Dp, is catalytically active and can be expressed atlevels 10-fold higher than that normally required toproduce the PtdEtn required for cell growth. The catalyticactivity of Psd2-C2Dp can be measured using a 1-acyl,2-(NBD-aminocaproyl)-PtdSer substrate that spon-taneously inserts into membranes harboring the enzyme.Further analysis reveals that the Psd2-C2Dp undergoesthe same post-translational processing as its wild-typecounterpart and is localized correctly within the cell.Despite the high catalytic activity of the Psd2-C2Dpexpressing strains, the cells fail to grow unless they aresupplemented with Etn. Thus, the C2 domain of Psd2p hasan important non-catalytic role in the action of theprotein. Measurement of PtdSer transport in intact andpermeabilized cells reveals that this lipid cannot bedelivered to the enzyme lacking the C2 domain. Collec-tively, these results define a direct role for the C2 domainof Psd2p in the transport process. The mechanism ofhow the C2 domain is functioning remains to beelucidated. It could form a docking module that recognizesanionic phospholipids (e.g. PtdSer, PtdIns, PtdIns4P,PtdIns(4,5)P2) and perhaps proteins on the donor mem-brane. If the C2 domain only acts in docking, then otherproteins would need to contribute to the assembly of atransport apparatus for moving PtdSer to the acceptormembrane. Alternatively, the C2 domain might bind toand physically participate in the transport of the lipidbetween the membranes. With either mechanism it seemsprobable that Psd2p can function in concert with otherproteins present on the acceptor membrane to form aPtdSer transport complex.

    The genetic screening of the PSTB pathway identifiedanother mutant strain (pstB2), and its corresponding gene(PSTB2) and encoded protein (PstB2p), as a component inthe process of PtdSer transport to the locus of Psd2p [19].The mutant strain displays a profound defect in the

    transport dependent metabolism of PtdSer to PtdEtn inintact and permeabilized cells and reconstitution

    www.sciencedirect.comreactions with isolated organelles. The PstB2p is amember of the phospholipid exchange and transferprotein family and is structurally similar to Sec14p, aPtdIns and PtdCho transfer protein that participates inprotein trafficking events [47]. The gene complementingthe pstB2 mutant strain has been named PDR17(NP_014135) and SFH4 in other screens [48,49] but forconsistency it is referred to as PSTB2 in this review. ThePstB2p transfers PtdIns between liposomes and mito-chondria in cell-free assays but does not transfer PtdSer.The protein is amphitropic and can be either soluble ormembrane bound in yeast and in insect cell expressionsystems. The membrane bound form of the protein ishighly resistant to removal by alteration of ionic con-ditions. Reconstitution assays with permeabilized cellsand organelle preparations demonstrate that the proteinmust be present on the acceptor membrane for transfer ofnascent PtdSer to Psd2p to occur [16]. The mechanism-of-action of PstB2p is still unknown and many outstandingquestions need to be resolved. The protein is capable ofbinding phosphatidylinositol (PtdIns) but it is not clearhow this relates to PtdSer transport. This lipid bindingcould account for the amphitropic properties of PsdB2pand it could facilitate interactions between donor andacceptor membranes. The requirement for PstB2p on theacceptor membranes also suggests that it interacts withspecific proteins (perhaps Psd2p) at that location but thishas not yet been demonstrated.

    Additional studies with the PSTB pathway haveprovided further insights into the role of lipids in theprocess of PtdSer transport. In an effort to define theseparate molecular requirements of the donor andacceptor membranes in lipid transport, a chemicallydefined donor membrane system was developed [50]. Thedefined donors exhibit some interesting and unexpectedcharacteristics. The first unusual property is thatmembranes with a relatively planar geometry madefrom large unilamellar vesicles (with diameters ofw400 nm) are superior donors compared with highlycurved membranes made from small unilamellar vesicles.A second unusual property is that PtdSer-rich donors arethe preferred substrate for transport. Pure PtdSer vesicles(400 nm diameter) transfer the lipid to acceptors at20-times the rate of vesicles containing 50% PtdSer and50% PtdCho. The decline in PtdSer transfer with changein liposome composition is exponential and, at 50%PtdSer,the transport reaction is negligible. Interestingly, phos-phatidic acid (PtdOH) and PtdIns4P can reverse theinhibition caused by surface dilution with lipids such asPtdCho. The PtdOH completely reverses the inhibition,whereas the PtdIns4P partially reverses the inhibition. Inreconstitution studies with chemically defined donors, thefidelity of the process to the properties of PtdSer transportobserved in vivo and in permeabilized cells, was cruciallytested using both pstB2D and psd2-C2D mutants. Theseexperiments function as important tests to rule outspurious fusion of chemically defined donors with theacceptor membranes. The results reveal that the recon-stitution of transport with chemically defined donors

    faithfully recapitulates the properties of PtdSer transportfound in both intact and permeabilized cells. These

  • olipid traffic

    ubiquitin.

    Review TRENDS in Biochemical Sciences Vol.30 No.7 July 2005402S100BS100B is an EF-hand domain-Ca2C binding protein that seems tofunction as a regulator of lipid transport machinery on mammalianmitochondria-associated membrane (MAM) and/or mitochondria.

    Phosphatidylinositol-4-kinasePhosphatidylinositol-4-kinase phosphorylates phosphatidylinositol toyield the product PtdIns4P. This modification can serve to markmembrane domains for interactions with specific binding proteins.Genetic and biochemical studies implicate the PtdIns4 kinase, Stt4p, inphospholipid transport. The PtdIns4P also functions as an importantprecursor to PtdIns(4,5)P2, which also acts as a membrane recognitiondomain for interaction with binding proteins. Cleavage ofBox 2. Emerging lipid and protein motifs implicated in phosph

    UbiquitinUbiquitin is a 76-amino acid protein that is attached to target proteinsvia an isopeptide linkage at specific lysine residues. In many cases,ubiquitin serves as a modification that destines the modified targetprotein for degradation via proteasomes. Recently, ubiquitin has beenrecognized in many systems to also serve as an important signalingmotif for either activating or inactivating proteins without degradation.This activation and inactivation can serve to regulate catalysis,transcription, subcellular trafficking and proteinprotein interactions.

    Ubiquitin ligasesUbiquitin ligases attach ubiquitin to target proteins. The ubiquitinligase subunit of interest in phospholipid traffic, Met30p, is part of alarge multiprotein complex. The role of Met30p is to bind to theholoenzyme and dictate the choice of target protein to be modified byfindings raise the possibility that segregated domains ofPtdSer, or PtdSer and PtdOH, or PtdSer and PtdIns4Pwithin the donor membrane, could serve as specific localregions for intermembrane lipid transport. Such domainscould arise as a consequence of specific proteins present inthe donor membrane that corral PtdSer and other anioniclipids. Alternatively, the anionic lipid-rich domains mightalso be induced in the donor membrane by interactionswith proteins in the acceptor membrane. Soluble adaptorproteins might also function to recognize specific lipid andprotein elements in donor and acceptor membranes. Thesemodels are all highly speculative at this stage but theyprovide a provocative hypothetical framework for futureexperiments.

    SummaryPhospholipid transport associated with new organelleformation is a fundamental process of biochemistry andcell biology whose mechanisms remain to be elucidated.Genetic and biochemical tools applied to the problem areimplicating specific proteins and lipids in the transportand its regulation, as summarized in Figure 3 and Box 2.The proteins or protein motifs implicated thus far includeubiquitin, ubiquitin ligase (Met30p), PtdIns4-kinase(Stt4p), a phospholipid binding protein (PstB2p), a solubleEF-hand protein (S100B) and a C2 domain (present onPsd2p). The lipids implicated in the process includePtdSer, PtdIns, PtdIns4P and PtdOH. PtdSer serves notonly as a substrate for transport and decarboxylation butalso as a potential ligand for the C2 domain of Psd2p.PtdIns, PtdIns4P and PtdOH are also potential

    www.sciencedirect.comPtdIns(4,5)P2 produces two important signaling molecules, diacylgly-cerol and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3).

    Phospholipid exchange and transfer proteinsPhospholipid exchange and transfer proteins can function toexchange lipids between membranes in vitro but their functionin vivo remains uncertain. The exchange protein described in thisreport, PstB2p, recognizes and transfers PtdIns in vitro. However,in vivo it is an essential component in PtdSer transport to Psd2p. Theproperty of PtdIns recognition might promote the attachment tomembranes in vivo, where it functions as part of a larger molecularcomplex that transports PtdSer between membranes.

    C2 domainsC2 domains are Ca2C- and phospholipids-binding domains involved inproteinlipid interactions. A C2 domain that is present on Psd2p is notrequired for catalysis but is necessary for the transfer of PtdSerbetween donor and acceptor membranes.

    PtdSer domainsReconstitution studies with liposomes as donor membranes demon-strate that PtdSer-rich domains are the preferred source of the lipid fortransfer to Psd2p. Dilution of pure PtdSer domains with small amountsof PtdCho dramatically inhibits intermembrane transport of the lipid.

    Acidic phospholipidsReconstitution studies show that dilution of PtdSer domains withphosphatidic acid does not disrupt transport like PtdCho but has astimulatory effect on the process. PtdIns4P, the product of Stt4p,shows similar albeit weaker activity than phosphatidic acid.recognition ligands for docking reaction between thedonor and acceptor membranes. In addition, the anioniclipids could also function as nucleation sites for inter-action with membrane proteins either in their resident orapposed membranes that can serve to assemble dockingand transport machinery at regions of membrane contact.These molecules are likely to constitute an early list ofcomponents involved in non-vesicular polar lipid trans-port between organelles. From this current list ofmolecular participants, the focus now needs to movetowards defining their precise roles in the transportreactions by addressing the questions: (i) what interactswith what? (ii) Do proteinprotein and proteinlipidinteractions between membranes introduce physicalchanges in local membrane environments (e.g. segre-gation of PtdSer) that are essential for transport? (iii) Howare intermembrane docking and phospholipid transportinterrupted to stop the process and disengage the donorsfrom the acceptors? (iv) How applicable are the molecularmotifs used for aminophospholipid transport to othertypes of polar lipids? (v) Do mammalian cells use the samemolecular motifs for aminophospholipid transport asthose uncovered in yeast? These current, challengingquestions require new genetic, biochemical and physicalapproaches to the problem. The resolution of these issuesis likely to provide new insights into the mechanisms ofphospholipid transport during membrane biogenesis.

    AcknowledgementsThis work was supported by a grant from the National Institutes ofHealth 2R37 GM 32453.

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    Bridging gaps in phospholipid transportIntroductionOrganelle specific metabolism of aminoglycerophospholipidsGenetic screens for phospholipid transport mutantsBiochemical studiesStudies with transport mutantsSummaryAcknowledgementsReferences