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

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    N

    CHCH

    3

    CH

    CH

    H2C

    HC

    H C

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    O

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    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

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    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...