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. Author manuscript; available in PMC: 2020 Aug 1.
Published in final edited form as: Curr Opin Cell Biol. 2019 Apr 28;59:58–72. doi: 10.1016/j.ceb.2019.03.012

An equal opportunity collaboration between lipid metabolism and proteins in the control of membrane trafficking in the trans-Golgi and endosomal systems

Yaxi Wang 1, Carl J Mousley 2, Marta G Lete 3, Vytas A Bankaitis 1,3
PMCID: PMC7198063  NIHMSID: NIHMS1525884  PMID: 31039522

Abstract

Recent years have witnessed the evolution of the cell biology of lipids into an extremely active area of investigation. Deciphering the involvement of lipid metabolism and lipid signaling in membrane trafficking pathways defines a major nexus of contemporary experimental activity on this front. Significant effort in that direction is invested in understanding the trans-Golgi network/endosomal system where unambiguous connections between membrane trafficking and inositol lipid and phosphatidylcholine metabolism were first discovered. However, powered by new advances in contemporary cell biology, the march of science is rapidly expanding that window of inquiry to include ever more diverse arms of the lipid metabolome, and to include other compartments of the secretory pathway as well.

Introduction

The endomembrane systems that define the organelles of the eukaryotic secretory pathway are remarkably dynamic structures. The landmark studies that unraveled the biochemical mechanism of membrane trafficking focused on the contributions of proteins in the membrane dynamics of the eukaryotic secretory pathway, and protein-centric views understandably dominated the early discussions [1–3]. In the nearly thirty years since the first convincing demonstrations that lipids and appropriately coordinated lipid metabolism are essential contributors to Golgi membrane trafficking and vesicle fusion were reported [4–9], our view of secretory organelle dynamics has evolved dramatically. Much attention has been, and continues to be, invested in the analysis of phosphoinositides as regulators of membrane trafficking and as determinants of compartmental identity. As structurally distinct molecules that are modified at specific positions on the inositol ring, phosphoinositides write chemical barcodes on membrane surfaces that help platform compartment-specific biochemical reactions (Figure 1). New advances highlight how phosphoinositides, and particularly how phosphatidylinositol-4-phosphate, integrate other aspects of lipid metabolism into the larger design for membrane traffic control.

Figure 1.

Figure 1

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Phosphoinositides and their metabolism. Phosphoinositides are phosphorylated derivatives of phosphatidylinositol (PtdIns) and their production is catalyzed by distinct sets of lipid kinases that modify either the C3, C4 or C5 positions of the inositol headgroup. Reciprocally, phosphoinositides are dephosphorylated via the action of a cohort of phosphoinositide phosphatases. Whereas mammalian cells produce seven phosphoinositides [PtdIns 3-phosphate, PtdIns3P; PtdIns 4-phosphate, PtdIns(4)P; PtdIns-5-phosphate, PtdIns5P; PtdIns 3,5-bisphosphate, PtdIns(3,5)P2; PtdIns 4,5-bisphosphate PtdIns(4,5)P2; PtdIns 3,4-bisphosphate, PtdIns(3,4)P2; PtdIns 3,4,5-triphosphate, PtdIns(3,4,5)P3], most organisms produce fewer. For example, baker’s yeast (Saccharomyces cerevisiae) and plants produce the first five, but lack the capacity to produce PtdIns(3,4)P2 or PtdIns(3,4,5)P3. PtdIns(4)P metabolism is highlighted in this figure as this phosphoinositide is the focus of discussion.

Phosphoinositide distribution in organelles

With regard to phosphoinositides serving as membrane signaling platforms, early live cell lipid imaging studies were made possible by development of genetically encoded fluorescence biosensors with positional specificities for phosphorylated headgroups [10,11]. Initial studies with such biosensors reported compartment-specific enrichments for particular phosphoinositides. For example, the endocytic system typically scored as enriched in PtdIns(3)P, the TGN in PtdIns(4)P, the vacuole/lysosomal system in PtdIns(3,5)P2, and the plasma membrane in PtdIns(4,5)P2, PtdIns(3,4)P2 and PtdIns(3,4,5)P3. Those compartment-specific profiles gave birth to the view that phosphoinositides are primary determinants of compartmental identity [12].

While there is no doubt that specific phosphoinositides play important contributing roles as coincidence signals that help write a biochemical code for compartment identity, this concept is an oversimplification. That the phosphoinositide-binding biosensor domains register other factors in membranes than just the phosphoinositide ligands themselves was indicated by the results of cell phenotyping analyses where the biological consequences of biosensor overexpression were monitored [13,14•]. Phosphoinositide distributions are also a function of the model organism — suggesting a strict identity code is not a rigid principle. For example, plants do not follow the mammalian script in terms of the shape of organelle-specific enrichment of phosphoinositides. Biosensor imaging experiments suggest PtdIns(4)P is most highly represented in plant plasma membranes [14•] and enrichment of PtdIns(4,5)P2 is scored not only in plant plasma membranes but also in intracellular compartments (secretory or endocytic vesicles?) in the tip cytoplasm of growing root hairs [15,16].

Moreover, a clear demonstration that phosphoinositide distribution is less organelle specific than first advertised is highlighted by characterization of a PtdIns(4)P-specific probe (P4M) engineered from the SidM protein of the bacterial pathogen Legionella pneumophila. Consistent with the PtdIns(4)P distribution profiles previously described from immune-electron microscopy approaches [17], P4M-based biosensors detect PtdIns(4)P pools not only in mammalian late-Golgi compartments, but also in endosomes, lysosomes, and in the plasma membrane [18]. Those findings emphasize that phosphoinositide biosensor localization in cells cannot be confidently extrapolated to phosphoinositide mass. The biosensors only report phosphoinositide pools accessible to the specific biosensor being used, and we often do not understand the parameters that determine accessibility. This interpretive caveat applies to the use of biosensors for imaging the intracellular distribution and dynamics of any lipid.

Lipid signaling in the TGN/endosomal system

If one were tasked with monitoring the physiological status of a cell, one would be well-advised to place a stethoscope on the highly integrated system of trans-Golgi network (TGN) and endosomal compartments [19,20]. Collectively, this system consists of a continuum of maturing organelles that form a signaling node where membrane trafficking interfaces with multiple aspects of lipid metabolism. These aspects now include metabolism of glycerol-based phospholipids (e.g. phosphoinositides; phosphatidylcholine, PtdCho; phosphatidic acid, PtdOH; phosphatidylserine, PtdSer), neutral lipids (e.g. diacylglycerol, sterols), sphingolipids (ceramide, sphingomyelin), and lyso-phospholipids (lipids lacking one of the two acyl chains). The focus of this review will be on the interface between these various aspects of lipid metabolism and membrane trafficking in the TGN/endosomal system.

PtdIns(4)P production in the TGN/endosomal system

The available evidence identifies PtdIns(4)P signaling as a major nexus for integrating wider aspects of lipid metabolism with membrane trafficking through the TGN/endosomal system. This lipid is produced via the action of PtdIns 4-OH kinases (PI4K), and the pools of PtdIns(4)P that reside in different intracellular compartments are produced by distinct enzymes. Mammalian PI4Ks fall into two distinct structural classes categorized as type II enzymes (PI4KIIα, PI4KIIβ) and type III enzymes (PI4KIIIα, PI4KIIIβ) [21]. The yeast type II enzyme is Lsb6 whereas the yeast PI4KIIIα and PI4KIIIβ enzymes are designated Stt4 and Pik1, respectively [22–25]. The type II PI4Ks localize to the TGN/endosomal system, to post-Golgi vesicles and the plasma membrane [26–29], and perhaps to the endoplasmic reticulum [30,31]. The mammalian and yeast PI4KIIIα and PI4KIIIβ enzymes primarily localize to the plasma membrane and TGN/endosomal compartments, respectively [25,32].

The type II enzymes remain poorly characterized, but available evidence from mammalian, fly and yeast models indicates a role in the endosomal system [33–35]. Interestingly, the yeast type II PI4K (Lsb6) is neither essential for cell viability, nor does its recognized involvement in stimulating endosome dynamics require PI4K enzymatic activity [35]. In neither the mammalian nor the fly cases is it firmly established whether the endosomal dynamics phenotypes require type II PI4K lipid kinase activities, or not. By contrast, the type III proteins are each individually essential for cell viability, collectively account for >90% of the PtdIns(4)P synthesis in yeast cells, and these enzymes are non-redundant in terms of biological function [24]. This biological non-redundancy reflects the functional distinction of the respective PtdIns (4)P pools produced. The plasma membrane-localized PI4KIIIα generates plasma membrane pools of the phosphoinositide, whereas the TGN/endosome-localized PI4KIIIβ is responsible for producing TGN/endosomal pools of PtdIns(4)P [24,36,37].

In terms of membrane trafficking through the TGN/endosomal system, PtdIns(4)P pools produced by the type IIIβ otholog Pik1 are of primary importance. Direct evidence to that effect comes from demonstrations that impaired Pik1 PtdIns 4-OH kinase activity evokes a defect in protein export from late Golgi compartments in yeast [38,39]. Our current understanding for how PtdIns(4)P signaling regulates TGN/endosomal membrane trafficking functions rests on identification of PtdIns(4)P effectors that translate the PtdIns(4)P chemical code to action at the level of membrane trafficking. PtdIns(4)P effectors include clathrin adaptors [40,41], and other cargo adaptors such as the yeast Vps74 PtdIns(4)P binding protein and its mammalian ortholog GOLPH3 [42–45]. GOLPH3 is proposed to represent a core component of the TGN membrane trafficking machinery that interacts with the non-conventional actin-based myosin Myo18A to force membrane scission and release of TGN-derived transport vesicles [44].

The interface of PtdIns(4)P signaling with activities of membrane trafficking regulators such as Rab and Arf family GTPases is also well-established. It includes interactions with Rab nucleotide exchange factors and tethers [46–48], and with proteins that specifically bind GTP-bound Arf–GTP. Those include the Gga proteins [49–51], the ‘four phosphate adaptor proteins’ (FAPPs) that interact with both ARF-GTP and with PtdIns(4)P [52,53], and protein kinase D (PKD) that collectively contribute to the biogenesis of Golgi-derived transport carriers [54,55].

PtdIns(4)P and broader regulation of lipid metabolism in the TGN/endosomal system

Initial studies demonstrated that trafficking from the yeast TGN required a proper coordination of PtdCho biosynthesis with essential membrane trafficking pathways, and that the threshold requirement of PtdIns(4)P for trafficking from the TGN/endosomal system is powerfully modulated by metabolic flux through the CDP-choline pathway for PtdCho biosynthesis [5,6,56]. Those findings show that functional coordination of lipid metabolic pathways with phosphoinositide signaling is a foundational concept underlying control of TGN membrane trafficking. We now appreciate that PtdIns(4)P signaling coordinates the activities of other aspects of lipid metabolism in the TGN/endosomal system, and that it does so in a manner that modulates membrane trafficking functions as well. Significant focus in this regard is shifting to the various involvements of sphingolipids and cholesterol in traffic control (Figure 2).

Figure 2.

Figure 2

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PtdIns(4)P and sphingolipid metabolism are coordinated in the TGN/endosomal system. Arf1-mediated recruitment of PKD to TGN membranes, and activation of the enzyme, requires diacylglcerol which itself is generated by sphingomyelin synthase (SMS) that consumes ceramide (chaperoned by CERT) and PtdCho to produce sphingomyelin (SM) and diacylglycerol (DAG). CERT and PI4KIIIβ are both PKD substrates. PKD downregulates the former and activates the latter, thereby providing a mechanism for both sensing and coordinating the activities of sphingolipid and PIP metabolic pathways in the TGN. Vesicle biogenesis can be generated by lateral segregation of sphingomyelin into microdomains that platform vesicle formation and perhaps clustering of cargo receptors such as those of the p24 family and Cab45 proteins, by the action of the PtdIns(4)P-binding protein FAPP2 which tubulates membranes and also regulates glucosylceramide (GlcCer) metabolism in the Golgi to promote complex GSL synthesis, and by the action of PtdIns(4)P in recruiting various cargo adaptors or ‘receptors’ that promote cargo packaging into forming vesicles.

One case in point involves the linkage of TGN membrane tubulation and vesicle biogenesis catalyzed by FAPP2 with integration of the metabolism of sphingolipids such as sphingomyelin, glucosylceramide and higher-order glyco-sphingolipids. FAPP2 is both a PtdIns(4)P-binding and a glucosylceramide-binding protein that regulates complex glycolipid synthesis in the Golgi system [52,57]. Ceramide transfer protein (CERT) is a PtdIns(4)P-binding protein required for sphingomyelin synthesis in Golgi membranes, and CERT is suggested to mediate non-vesicular transfer of ceramide from the endoplasmic reticulum to TGN membranes [58,59].

Sphingolipid metabolism and TGN PtdIns(4)P status are tightly coordinated [60••]. Specifically, the net efficiency by which the Sac1 PIP-phosphatase degrades PtdIns4(P) in the mammalian TGN is inversely proportional to metabolic flux through the SL-biosynthetic pathway in Golgi membranes. This metabolic coordination acts as a rheostat for maintaining TGN sphingolipid content in the face of sphingolipid flow into and from the organelle. This rheostat interfaces with membrane traffic because sphingomyelin (and other sphingolipids) can laterally organize themselves with cholesterol to form microdomains that provide a chemical environment that platforms formation of distinct classes of transport carriers into which transport cargo can be differentially sorted [61,62,63•]. In what might prove to be a related mechanism, the biological function of some cargo receptors, such as Cab45, is potentiated by the sphingolipid status of TGN membranes [64••].

Another example is provided by the yeast peripheral PtdIns(4)P-binding protein Vps74 whose function is responsive to both sphingolipid and PtdIns(4)P signals. Vps74 functions in retrograde transport of integral membrane glycosyltransferases from TGN to medial Golgi compartments, and the PtdIns(4)P pool involved in Vps74 recruitment to the TGN surface is produced by the yeast PI4KIIIβ enzyme Pik1 [42]. Yeast cells lacking Vps74 not only show deficits in specific Golgi sphingolipids, but show broadened PtdIns(4)P distribution throughout the Golgi system [65]. While Vps74-deficient cells are viable, these mutants require complex sphingolipid synthesis to maintain viability, and mutants compromised for complex sphingolipid production show deficits in glycosyl-transferase retrieval back to medial Golgi compartments. This is likely a conserved pathway. Rotini (Rti), the Drosophila Vps74 ortholog, also functions in retrieval of a special class of glycotransferases from the TGN to medial Golgi, and these particular glycosyltransferases are required for correct processing of heparan sulfate proteoglycans [66,67].

Lipid microdomains are but one mechanism by which sphingolipids can modulate trafficking processes. These can also function as de facto cofactors where a single sphingomyelin molecule of a specific molecular species interacts directly with a protein component of the membrane trafficking machinery and modulates its activity. The foundational example is provided by the single transmembrane p24 protein whose activity in recruiting cargo for packaging into COP1-coated vesicles requires its transmembrane domain to bind to a sphingomyelin molecule [68]. Interestingly, the SM-binding signature of the p24 transmembrane domain (VXXTLXXIY) is present in the transmembrane domains of a number of other proteins as well [69].

PtdIns(4)P and regulation of PtdSer membrane asymmetry in the TGN/endosomal system

Topological distribution of specific phospholipids is also responsive to PtdIns(4)P signaling. A phosphatidylserine (PtdSer) flippase of the P4-type ATPase family (Drs2) functionally interacts with Arf-dependent and clathrin-dependent pathways for TGN-derived vesicle biogenesis in yeast [70]. Drs2 retrieves PtdSer from the lumenal leaflet of TGN/endosomal membranes to the cytoplasmic leaflet and establishes a leaflet asymmetry so that PtdSer is relatively enriched on the TGN/endosome surface. The Arabidopsis Drs2 ortholog Ala3 functions similarly in coupling PtdSer flippase activity to membrane trafficking from TGN/endosomes [71].

In terms of mechanism, the ATPase and PtdSer flippase activities of Drs2 are stimulated by binding to both PtdIns (4)P generated by the PI4KIIIβ Pik1 and the Gea2 Arf guanine nucleotide exchange factor. Thus, PtdIns(4)P and the protein activator of Arf-GTP production cooperate in a coincidence detection mechanism to regulate Drs2 activity in TGN membranes [72]. Drs2-generated PtdSer asymmetry supports formation of TGN-derived clathrin coated vesicles by increasing local membrane curvature and negative charge so that the Arf GTPase activating protein Gcs1 can be recruited to TGN membranes to optimize vesicle formation and cargo loading [73]. Other evidence suggests that Drs2 activity controls lateral distribution of sterol in TGN membranes to regulate protein sorting into distinct classes of vesicles [74].

PITPs and biological specification of PtdIns(4)P signaling

How is PtdIns(4)P homeostasis controlled at the level of synthesis and at the level of degradation? With regard to the former, it is an underappreciated concept that both PI4KIIIα and PI4KIIIβ are intrinsically inefficient enzymes in biological contexts. That is, these enzymes, even as wild-type enzymes, are incapable of producing sufficient PtdIns(4)P to overcome the activities of erasers of PtdIns(4)P signaling to exert biological responses. Nature solves this problem for eukaryotic cells by the design of PtdIns transfer protein (PITPs) that potentiate PtdIns(4)P production by PI4Ks so that sufficient PtdIns (4)P is produced to support PtdIns(4)P signaling in the face of the opposing action of signaling antagonists [56,75,76]. Although the presently known PITPs fall into two ancient and distinct structural families [i.e. those with the Sec14 fold and those that adopt a StART (Steroidogenic Acute Regulatory Protein-related lipid transfer domain)-like fold], the available evidence indicates these PITPs operate on a similar mechanistic principle. That is, PITPs couple a heterotypic exchange cycle involving PtdIns and a second lipid ligand (classically PtdCho) to ‘present’ PtdIns to the PI4K. This heterotypic exchange cycle renders the engaged PtdIns molecule a superior substrate for the PI4K and thereby potentiates the catalytic activity of the enzyme [68,75–77].

The evolutionary design of PI4Ks as inefficient enzymes holds significant advantages. It not only allows cells to exert exquisite control over PI4K activity and PtdIns(4)P signaling, but it also allows the physical organization of PI4K signaling units into discrete nanocircuits (or signaling pixels) assembled around individual PI4K enzyme complexes [56,76]. High resolution ‘pixelation’ of membrane surfaces provides an attractive mechanism for functionally expanding what is a chemically homogenous PtdIns(4)P code into a high-resolution membrane screen capable of supporting a diverse set of biological outcomes for PtdIns(4)P signaling [4,75,78,79]. PITP-mediated instructive channeling of PtdIns(4)P synthesis and signaling to specific biological outcomes provides new opportunities for generating small molecule inhibitors that have the property of imposing PtdIns(4) P pool-specific interference on signaling [80–82].

PITP-mediated control of TGN/endosomal PtdIns(4)P in mammalian systems

The biological importance of PITPs is on display in vertebrate (zebrafish) and mammalian (mouse) contexts as well and, in all cases studied to date, the ability of the PITP to stimulate PtdIns(4)P production is required for biological activity [76,77]. More recent work with START-like PITPs connects Golgi PI4K signaling with establishment and/or maintenance of extreme cell polarity in embryonic neural stem cells (NSCs) of developing mammalian forebrain (Figure 3). A pair of START-like PtdIns/PtdCho-exchange PITPs (PITPα and PITPβ) support production of a TGN/endosomal PtdIns(4)P pool sufficient for recruitment of GOLPH3 and CERT in a cell-autonomous pathway essential for: (i) polarized distribution of the Golgi system and NSC polarity, and (ii) non-cell-autonomous maintenance of the neurogenic niche that supports development of the neocortex [83••]. Drosophila encodes one PITPα/PITPβ ortholog (Vib) and, similar to the case of embryonic mammalian NSCs, deficiencies in this activity impair fly neuroblast homeostasis. In flies, Vib supports production of a plasma membrane PtdIns(4)P pool (likely in cooperation with PI4KIIIα) to facilitate a non-muscle myosin and actin-based asymmetric partitioning of cell fate determination factors during cytokinesis [84••].

Figure 3.

Figure 3

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Class I START PITP function in apical loading of the Golgi system in neural stem cells. Class I START PITPs exchange PtdCho and PtdIns, thereby stimulating PtdIns 4-OH kinase activity on late Golgi membranes. The PtdIns(4)P pool recruits GOLPH3 and CERT to Golgi membranes with GOLPH3 subsequently engaging the apically directed actin machinery via the nonconventional myosin Myo18A. This interaction promotes loading of the Golgi system to the neural stem cell apical process, and in this fashion produces an asymmetry critical for establishment/maintenance of NSC polarity. CERT functions either downstream of GOLPH3 or in a parallel pathway to regulate Golgi positioning and NSC polarity. The underlying mechanism remains under investigation and might involve regulation of apically directed, rather than bulk, membrane trafficking.

GOLPH3, the START-like PITP Pitpnc1, and cancer

GOLPH3 not only binds PtdIns(4)P in the TGN/endosomes of mammalian NSCs, but there exists a compelling cancer link between GOLPH3 and yet another START-like PITP designated PITPnc1 [85,86••]. In that regard, PITPnc1 expression is downregulated by the ‘anti-metastatic’micro-RNA miR-126 whose silencing results in enhanced angiogenesis and aggressive tumor metastasis. PITPnc1 likely contributes to production of the TGN PtdIns(4)P pool required for membrane recruitment of GOLPH3 in cancer cells, and is itself reported to be a PtdIns(4)P binding protein — an activity that might help recruit this PITP to TGN membranes via a positive feedback loop which powers recruitment of GOLPH3 and the RAB1B GTPase to enhance secretory capacity of the TGN/endosomal system ([86••]; Figure 3). A GOLPH3/RAB1 connection is also evident in Drosophila where these components cooperate to support membrane incorporation at the point of cleavage furrow ingression during cytokinesis [87].

Erasers of PtdIns(4)P signaling — the Sac1 phosphatase

With regard to mechanisms of downregulation of PtdIns (4)P signaling, key players were first recognized in the ‘bypass Sec14’ genetic screen that identified loss-of-function mutations that restored cell viability (and efficient membrane trafficking through the TGN/endosomal system) to yeast cells completely lacking Sec14 PITP function. Those ‘bypass Sec14’ mutants fall into two general classes: (i) one where PtdIns(4)P levels are dramatically elevated, and (ii) another where mutants thrive in the face of low PtdIns(4)P levels normally insufficient to support either membrane trafficking from TGN/endosomes or cell viability [5,56,88,89].

The first class identifies the Sac1 lipid phosphatase that is the major enzyme for degrading PtdIns(4)P in the cell [90,91]. Its primary compartment of residence is the endoplasmic reticulum (ER; 88,92), and this localization gave rise to proposals that Sac1 acts in trans at ER-plasma membrane contact sites to hydrolyze plasma membrane PtdIns(4)P pools [93]. Structural studies contradict this notion. Sac1 is built for degrading PtdIns(4)P in its membrane of residence (i.e. in cis; 94,95). Second, in addition to first reports of Golgi pools of Sac1 [88], other data indicate this lipid phosphatase cycles from ER to Golgi membranes in response to nutrient-limiting or growth factor-limiting conditions [96,97]. Thus, Golgi PtdIns(4)P-dependent secretory activity can be controlled by regulating Golgi Sac1 pools.

In yet a third line of evidence, yeast Vps74 binds to both PtdIns(4)P and to Sac1, and has the capacity to locally control PtdIns(4)P pools [65]. In what is likely a constitutive process, Vps74 encounters Sac1 that has trafficked from the ER to the Golgi, and this complex maintains the increasing PtdIns(4)P gradient observed as one moves from early to medial to late Golgi compartments. Sac1 egress from ER is regulated in part by a Sac1 phosphorylation/14-3-3 protein-binding cascade that promotes Sac1 packaging into COPII vesicles [98].

Erasers of PtdIns(4)P signaling — the Kes1 PtdIns(4)P/sterol exchange protein

Included in the second class of ‘bypass Sec14’ mutants are loss-of-function mutations in a member of the oxysterol binding protein family Kes1/Osh4 [89]. Of the seven yeast oxysterol binding related proteins (ORPs or Osh proteins), Kes1/Osh4 is unique in its functional antagonism of Sec14-dependent PtdIns(4)P signaling [89,99••].Curiously, like the PtdIns(4)P signaling protagonist Sec14, Kes1 is also a lipid transfer protein in vitro — in this case one that executes a heterotypic PtdIns(4)P/ergosterol exchange reaction and functions as a negative regulator of PI4KIIIβ (Pik1)-dependent PtdIns(4)P signaling and membrane trafficking through the TGN/endosomal system [100,101]. Surprisingly, while Kes1 in vivo activity as PtdIns(4)P signaling antagonist in the TGN/endosomal trafficking program is fully ablated by defects in PtdIns(4)P binding, that same activity is enhanced by sterol binding deficits [102].

There are two competing ideas for how Kes1/Osh4 executes its biological function. One proposes an inter-organelle lipid transfer mechanism, while the second posits a sterol-based tuning mechanism that determines the amplitude of PtdIns(4)P sequestration by Kes1 from its pro-trafficking effectors (Figure 4). The first is the counter-current hypothesis where Kes1/Osh4 is envisioned to transfer sterol from ER to TGN membranes, and then reciprocally transfer PtdIns(4)P from the TGN to the ER for Sac1-mediated degradation [103,104]. This general idea has been expanded to ORPs in general, and to other inferred non-vesicular pathways for inter-organelle lipid transfer. Included in that extension is mobilization of PtdSer to the plasma membrane with a return itinerary for PtdIns(4)P back to the ER for Sac1-mediated hydrolysis — although there is some controversy on that point regarding which phosphoinositides and even which lipids are transported in counter-current fashion [105,106,107•,108••].

Figure 4.

Figure 4

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Models for how Kes1/Osh4 downregulate PtdIns(4)P signaling in the TGN. Left panel: The sterol/PtdIns(4)P counter-current hypothesis. This model proposes the sterol/PtdIns(4)P exchange reaction occurs on TGN membranes to supply the system with sterol transported from the ER, and ferries PtdIns(4)P from the TGN to ER for its degradation in that compartment by the Sac1 lipid phosphatase. The degradation step is proposed to provide the energy to drive the cycle. In this scenario, both sterol transport to the TGN and PtdIns(4)P counter-transport to the ER are essential activities for Kes1/Osh4 biological function. Right panel: The sterol/sensing hypothesis. This model posits the operative sterol/PtdIns (4)P exchange reaction occurs on TGN membranes and does not involve inter-organelle lipid transport as functional prerequisite. The competition for Kes1/Osh4 binding to these lipids coordinates sterol status (scored by sterol binding) with the strength of Kes1/Osh4-mediated sequestration of PtdIns(4)P from its pro-trafficking effectors on TGN membranes (scored by PtdIns(4)P-binding). The basic principle is that Kes1/Osh sets a trafficking brake upon binding PtdIns(4)P, and lipid exchange with an available sterol molecule displaces a bound PtdIns(4)P molecule from Kes1/Osh4 – thereby releasing the signaling/trafficking brake by releasing sterol-bound Kes1/Osh4 from TGN membranes. Thus, the fraction of Kes1/Osh4 on TGN membranes reflects the strength of the signaling/trafficking brake, and this scalar is indirectly proportional to the availability of sterol for binding by Kes1/Osh4. This model predicts Kes1/Osh4 lacking sterol binding activity is a powerful signaling brake (enhanced function), while Kes1/Osh4 defective in PtdIns(4)P binding is bereft of biological function.

There remains considerable debate regarding other experimental foundations of the counter-current model as well. A recent study in mammalian cells suggests as much as 50% of the cellular PtdIns(4)P is turned over in the Golgi system via the OSBP/Sac1 counter-current pathway as a function of the size of the sterol pool to be mobilized from the ER [109••]. In contradiction to that report, other evidence from mammalian cells shows regulation of PtdIns(4)P and cholesterol homeostasis by that very same mammalian OSBP is substantially (if not totally) independent of Sac1 activity [110••]. The base claim that yeast and mammalian ORPs are lipid carriers that promote non-vesicular sterol trafficking between intracellular membranes is also a controversial one. Some studies argue in favor [111,112], others against [113,114,115••]. However, the fact that neither ablation of all known membrane tethers that might support nonvesicular lipid transfer through contact sites, nor wholesale ablation of ORP function, has measurable effect on sterol transport in yeast makes a powerful case against the idea that ORPs are authentic sterol transporters – through contact sites or otherwise [115••].

Unlike the indirect assays typically used to infer non-vesicular lipid transport in living cells, the Sec14/Kes1 functional antagonism at the level of PtdIns(4)P signaling in the yeast TGN provides a physiologically relevant assay for Kes1/Osh4 function — one supported by an unambiguous biological phenotype. It is in this context that key predictions of the counter-current hypothesis can be examined with greater confidence. While the prediction of the model that PtdIns(4)P binding is an essential functional property of Kes1/Osh4 is fulfilled [99••,100,101], other key predictions are not supported by experiment. Contrary to the prediction that sterol binding is required for Kes1/Osh4 activity in vivo as a sterol carrier, Kes1 activity as an antagonist of PtdIns(4)P signaling at the TGN is enhanced upon loss of its ability to bind sterol [102]. Moreover, the role of the Sac1 PtdIns(4)P phosphatase as proposed by the counter-current model also does not easily conform to in vivo data. Whereas Kes1/Osh4 and Sac1 deficits have in common the ‘bypass Sec14’ phenotype, there is a curious PtdIns(4)P pool discrepancy with the basic foundation of the counter-current hypothesis. Kes1/Osh4 is responsive to the Pik1 (PI4KIIIβ)-generated pool of PtdIns(4)P in TGN/endosomes [100]. Yet, the cellular pool of PtdIns(4)P degraded by Sac1 in vivo is almost exclusively restricted to that produced by Stt4 (PI4KIIIα; 24,116). Arguments that these discrepancies can be accounted for by functional redundancy of the six other ORPs with Kes1/Osh4 is contra-indicated by experiment. None of the remaining ORPs – either individually or in combination – execute Kes1/Osh4 functions in the TGN [89,99••,102].

The alternative hypothesis proposes what is fundamentally a sterol sensing mechanism [102]. This model posits Kes1/Osh4 functions as a sensor which employs its differential lipid binding activities to run a heterotypic sterol/PtdIns(4)P exchange cycle on TGN membranes. This exchange cycle contributes to a rheostat mechanism that coordinates amplitude of the Kes1/Osh4 brake on PtdIns(4)P signaling and trafficking in response to ‘available’ sterol (Figure 4). This model is compatible with the existence of a TGN/endosomal Sac1 pool that degrades PtdIns(4)P locally, given the strength of any Kes1/Osh4 brake on PtdIns(4)P-signaling would be sensitive to Sac1 activity, and with the experimental result that loss of sterol binding capacity further activates Kes1/Osh4. How generally applicable this model is to the functional mechanisms of other ORPs remains to be determined.

New frontiers at the TGN/endosomal membrane trafficking/lipid signaling interface

That there is more to be discovered regarding PtdIns(4)P signaling in membrane traffic control is highlighted by a remarkable study that identifies a requirement for a Vps13/centrin complex on both TGN and late endosomal membranes for efficient membrane trafficking from the former compartment to the latter [117••]. The novelty here is that centrin is a core component of the spindle pole body that organizes the mitotic spindle in dividing cells. Further evidence to indicate a direct role for the Vps13/centrin complex in lipid signaling comes from studies of sporulating yeast cells that leverage PtdOH production via phospholipase D activity to build new membrane envelopes (prospore membranes) that encapsulate the newly replicated genomes produced by meiosis in the nutrient-deprived cells [118,119]. Prospore expansion is impaired in Vps13-deficient cells and this defect is associated with diminution of PtdOH and PtdIns(4)P pools. Congruent conclusions are reached in a mammalian cell model [120].

Taken together, the data identify the highly conserved Vps13 as yet another integrator for PtdIns(4)P, and perhaps PtdOH, signaling with TGN/endosomal membrane trafficking. The biological relevance of these findings is highlighted by the fact that inherited neurodegenerative diseases are associated with mutations in three of the four Vps13 isoforms expressed in humans [121]. On the basis of structural analyses and lipid transfer assays in vitro, it is suggested that Vps13 isoforms transfer lipids between organelles in vitro via a non-vesicular mechanism [122•].

Membrane trafficking and lyso-phospholipids

Active lipid metabolism in membranes both produces and consumes lipid precursors, or degradation products and modulates their ability to undergo dynamic processes such as vesiculation and tubulation. These intermediate metabolites do so in part by their disruption of membrane packing order and resultant enhancements of membrane malleability [123]. The Lands cycle of lipid deacylation/reacylation is one such a metabolic activity, and phospholipases A2 and the lyso-PtdOH acytransferase LPAAT3 cooperate to control Golgi dynamics and membrane trafficking [124,125].

New progress now elevates the rather sparsely investigated arena of acyl chain remodeling and lyso-phospholipid metabolism onto the main stage of lipid-mediated regulation of Golgi secretory function. With regard to the former, a protein complex that includes PI4KIIIβ catalyzes reacylation of lyso-PtdOH to produce of a pool of PtdOH that helps drive the membrane fission reaction that results in release of Golgi-derived vesicles [126•]. The demonstration that fatty-acid binding protein 5 (FABP5) modulates the kinetics of the Sar1 GTPase cycle, and promotes the biogenesis of COPII vesicles suitable for packaging of large cargo, might well reflect an involvement for the Lands cycle in that process [127•]. With regard to lyso-phospholipids, there is new evidence for a contribution of lyso-phosphatidylinositol to formation of COPII vesicles on the ER surface [128••]. While those findings were gleaned from a rather complex genetically sensitized yeast model, the general concept is likely to apply more broadly. A phospholipase A1 that converts PtdOH into lyso-PtdOH (i.e. by cleaving fatty acid from the sn-1 position) is a structural component of mammalian ER exit sites [129].

Global aspects of TGN/endosomal PtdIns(4)P signaling

An exciting new frontier is emerging — namely, the surprising linkage of TGN/endosomal lipid signaling with stage-specific regulation of cell cycle progression. One area in this new frontier involves PtdIns(4)P signaling and the mitotic spindle. This connection was alluded to above regarding the role of Vps13 and PtdIns(4)P in regulating membrane trafficking through the TGN/endosomal pathway in a mechanism that shows an obligate requirement for Vps13 binding to the spindle pole body component centrin to execute the process [117••]. The curious relationship between the Golgi system and the mitotic spindle is also on display in mammalian cells with diminished Sac1 PtdIns(4)-phosphatase activity. In that case, the Golgi is fragmented (but competent for bulk membrane trafficking) and the mitotic spindle is deranged with failure to progress through G2/M and resolve into a productive cell division [130].

Another direct linkage of chromosome metabolism to Golgi PtdIns(4)P signaling comes from the demonstration that the mammalian TGN/endosomal PtdIns(4)P effector GOLPH3 is phosphorylated by a DNA-damage-activated-protein-kinase. This modification results in fragmentation of the Golgi ribbon, as a result of enhanced interaction of GOLPH3 with Myo18A, and an accompanying membrane trafficking block [131]. Genetic studies in yeast further support the existence of a TGN/chromosome integrity checkpoint by showing that activity of the GOLPH3 ortholog Vps74 is required for prevention of telomere shortening, and that this level of control is likely imposed at the level of telomere capping [132].

The yin and yang of Sec14-mediated potentiated PI4KIIIβ signaling and Kes1/Osh4-mediated downregulation of PtdIns(4)P-signaling in the TGN/endosomal system provides another intriguing example where a membrane trafficking control circuit extends to cell cycle contexts [99••,102]. Kes1/Osh4 is not only a brake on membrane trafficking through the TGN/endosomal system, but it is also a physiologically relevant brake on PtdIns(4)P-dependent progression through the G1 stage of the cell cycle (Figure 4). The mechanistic basis for this activity is the reciprocal relationship between Kes1/Osh4 action and mTOR activity [102]. Moreover, a role for Kes1/Osh4 in promoting cell aging and shortening replicative lifespan is also indicated [99••].

The importance of Kes1/Osh4 as central integrator of TGN/endosomal PtdIns(4)P signaling with membrane trafficking and with the cell cycle is further emphasized by the demonstration that Kes1/Osh4 is a major non-histone target for the highly conserved NuA4 lysine acetyltransferase. NuA4 directly regulates Kes1/Osh4 PtdIns(4)P-binding by acetylating a key Lys residue involved in coordinating PtdIns(4)P headgroup binding [99••]. The base concept that cell cycle functions are regulated by lipid metabolism in the endosomal system is further reinforced by the demonstration that a late endosomal pool of PtdIns(3,4)P2 inhibits mTOR activation [133••].

Concluding remarks

We live in a fast pace era in the field of lipid cell biology — particularly as it relates to membrane traffic control. The field holds tremendous excitement as shown by the rapid progress with which fundamental advances are being made in the arena. However, a number of fundamental mechanistic questions remain under serious debate, and technical obstacles to further progress also exist and need to be overcome. Reliable functional assays that directly report on protein activity in a firm physiological context need to be developed. It is our opinion that a number of the ongoing controversies are the result of conclusions being based on inferences gleaned from indirect readouts of unclear physiological significance. We anticipate model organism systems will continue to play an ever larger role in clarifying direction. Biosensors that reliably report on membrane properties such as surface curvature, surface electrostatics, fluidity, and lateral tension also need to be either developed or improved. These biophysical parameters must be key contributors to the regulation of lipid signaling and we presently do not have a suitable handle on how to assess those contributions.

The questions and technical challenges that confront the field notwithstanding, there is no longer any doubt that lipid signaling is a crucial component of the membrane dynamics program of the eukaryotic cell. This point is further reinforced by progress in understanding the key involvements of lipids in cellular membrane trafficking systems not discussed here. These include: (i) the fusion/fission cycle of lysosomes [134], (ii) the maturation of the phosphoinositide content of clathrin-coated vesicles as these structures progress in their trafficking itinerary through the endocytic pathway [135], (iii) the conversion of PtdIns(3)P to PtdIns(4)P pools for egress of membrane vesicles from endosomes into the exocytic pathway [136], and the fission/fusion cycle of autophagosomes [137]. Indeed, the breadth of the lipid metabolism/membrane trafficking interface is of such magnitude that it is reasonable to ask whether there is any such thing as a lipid metabolic pathway that executes solely a house-keeping function. The time has come to consider lipids and lipid metabolism on an equal footing with proteins when considering the mechanisms that lay the foundation for membrane traffic design.

Acknowledgements

We apologize to our colleagues whose important contributions were not included due to space restrictions. Work in the authors’ laboratory is supported by N.I.H. grants GM44530 and GM112591, and an award from the Robert A. Welch Foundation (BE-0017), to VAB.

Footnotes

Conflict of interest statement

Nothing declared.

References and recommended reading

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• of special interest

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