Ether lipids influence cancer cell fate by modulating iron uptake.

Cancer cell fate has been widely ascribed to mutational changes within protein-coding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with a high metastatic ability and cancer stem cell-like traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, leading directly to significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro three-dimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

Phosphoglycerate kinase 1 acts as a cargo adaptor to promote EGFR transport to the lysosome.

The epidermal growth factor receptor (EGFR) plays important roles in multiple cellular events, including growth, differentiation, and motility. A major mechanism of downregulating EGFR function involves its endocytic transport to the lysosome. Sorting of proteins into intracellular pathways involves cargo adaptors recognizing sorting signals on cargo proteins. A dileucine-based sorting signal has been identified previously for the sorting of endosomal EGFR to the lysosome, but a cargo adaptor that recognizes this signal remains unknown. Here, we find that phosphoglycerate kinase 1 (PGK1) is recruited to endosomal membrane upon its phosphorylation, where it binds to the dileucine sorting signal in EGFR to promote the lysosomal transport of this receptor. We also elucidate two mechanisms that act in concert to promote PGK1 recruitment to endosomal membrane, a lipid-based mechanism that involves phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and a protein-based mechanism that involves hepatocyte growth factor receptor substrate (Hrs). These findings reveal an unexpected function for a metabolic enzyme and advance the mechanistic understanding of how EGFR is transported to the lysosome.

GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis.

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.

Accurate electronic properties and non-linear optical response of two-dimensional MA2Z4.

Two-dimensional MA2Z4 (M = Mo, W, V, Nb, Ta, Ti, Zr, Hf, or Cr; A = Si or Ge; Z = N, P, or As) is a new lead in the 2D family, because it exhibits versatile properties by tuning the components M, A and Z. However, theoretical studies on MA2Z4 are quite limited, and electronic properties are mainly studied by standard DFT levels, which seriously underestimates the band gap. Here, we systematically investigated the electronic properties and nonlinear optical response of MA2Z4 using a hybrid HSE06 functional. It was found that replacing component Z changes the lattice constant most, while the lattice influence by component M substitution is only slight. We showed that the gap difference between PBE and HSE06 is generally about 30% but can be up to 101%. (MIV = Hf, Ti, or Zr)Si2N4 possesses multi-valley characteristics. Furthermore, the second-harmonic generation (SHG) responses of various MA2Z4 composites were also calculated. Three non-zero elements of second order non-linear susceptibilities are revealed for MA2Z4 with the relationship: d16 = d21 = d22, indicating that MA2Z4 belongs to the D3H1 space group. HfSi2N4 possesses a multi-valley characteristic, and exhibits the largest susceptibility under broad wavelengths and the value of d21 reaches 3697.04 pm V-1 at band gap resonance energy. Intriguingly, the non-linear coefficients of MoSi2P4 and MoSi2As4 in the IR region are two orders of magnitude larger than those of other well-known non-linear crystals, such as LiGaS2 and BaAl4S7. We further explored the anisotropic SHG response by the polar plot of intensity under different incident light into MA2Z4. Our work provides theoretical guidelines for further experimental explorations of MA2Z4 and paves the way for its utilization in non-linear optic devices.

Combined immunodeficiency due to a mutation in the γ1 subunit of the coat protein I complex.

The coat protein I (COPI) complex mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER). Five siblings with persistent bacterial and viral infections and defective humoral and cellular immunity had a homozygous p.K652E mutation in the γ1 subunit of COPI (γ1-COP). The mutation disrupts COPI binding to the KDEL receptor and impairs the retrieval of KDEL-bearing chaperones from the Golgi to the ER. Homozygous Copg1K652E mice had increased ER stress in activated T and B cells, poor antibody responses, and normal numbers of T cells that proliferated normally, but underwent increased apoptosis upon activation. Exposure of the mutants to pet store mice caused weight loss, lymphopenia, and defective T cell proliferation that recapitulated the findings in the patients. The ER stress-relieving agent tauroursodeoxycholic acid corrected the immune defects of the mutants and reversed the phenotype they acquired following exposure to pet store mice. This study establishes the role of γ1-COP in the ER retrieval of KDEL-bearing chaperones and thereby the importance of ER homeostasis in adaptive immunity.

The protein kinase Akt acts as a coat adaptor in endocytic recycling.

Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins1-3. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling4-6. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event.

ALDH7A1 inhibits the intracellular transport pathways during hypoxia and starvation to promote cellular energy homeostasis.

The aldehyde dehydrogenase (ALDH) family of metabolic enzymes converts aldehydes to carboxylates. Here, we find that the reductive consequence of ALDH7A1 activity, which generates NADH (nicotinamide adenine dinucleotide, reduced form) from NAD, underlies how ALDH7A1 coordinates a broad inhibition of the intracellular transport pathways. Studying vesicle formation by the Coat Protein I (COPI) complex, we elucidate that NADH generated by ALDH7A1 targets Brefeldin-A ADP-Ribosylated Substrate (BARS) to inhibit COPI vesicle fission. Moreover, defining a physiologic role for the broad transport inhibition exerted by ALDH7A1, we find that it acts to reduce energy consumption during hypoxia and starvation to promote cellular energy homeostasis. These findings advance the understanding of intracellular transport by revealing how the coordination of multiple pathways can be achieved, and also defining circumstances when such coordination is needed, as well as uncovering an unexpected way that NADH acts in cellular energetics.

The late stage of COPI vesicle fission requires shorter forms of phosphatidic acid and diacylglycerol.

Studies on vesicle formation by the Coat Protein I (COPI) complex have contributed to a basic understanding of how vesicular transport is initiated. Phosphatidic acid (PA) and diacylglycerol (DAG) have been found previously to be required for the fission stage of COPI vesicle formation. Here, we find that PA with varying lipid geometry can all promote early fission, but only PA with shortened acyl chains promotes late fission. Moreover, diacylglycerol (DAG) acts after PA in late fission, with this role of DAG also requiring shorter acyl chains. Further highlighting the importance of the short-chain lipid geometry for late fission, we find that shorter forms of PA and DAG promote the vesiculation ability of COPI fission factors. These findings advance a general understanding of how lipid geometry contributes to membrane deformation for vesicle fission, and also how proteins and lipids coordinate their actions in driving this process.

GAPDH inhibits intracellular pathways during starvation for cellular energy homeostasis.

Starvation poses a fundamental challenge to cell survival. Whereas the role of autophagy in promoting energy homeostasis in this setting has been extensively characterized1, other mechanisms are less well understood. Here we reveal that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibits coat protein I (COPI) transport by targeting a GTPase-activating protein (GAP) towards ADP-ribosylation factor 1 (ARF1) to suppress COPI vesicle fission. GAPDH inhibits multiple other transport pathways, also by targeting ARF GAPs. Further characterization suggests that this broad inhibition is activated by the cell during starvation to reduce energy consumption. These findings reveal a remarkable level of coordination among the intracellular transport pathways that underlies a critical mechanism of cellular energy homeostasis.

Reconstitution of COPI Vesicle and Tubule Formation.

The Golgi complex plays a central role in the intracellular sorting of proteins. Transport through the Golgi in the anterograde direction has been explained by cisternal maturation, while transport in the retrograde direction is attributed to vesicles formed by the coat protein I (COPI) complex. A more detailed understanding of how COPI acts in Golgi transport is being achieved in recent years, due in large part to a COPI reconstitution system. Through this approach, the mechanistic complexities of COPI vesicle formation are being elucidated. This approach has also uncovered a new mode of anterograde transport through the Golgi, which involves COPI tubules connecting the Golgi cisternae. We describe in this chapter the reconstitution of COPI vesicle and tubule formation from Golgi membrane.

Coordinated regulation of bidirectional COPI transport at the Golgi by CDC42.

The Golgi complex has a central role in the intracellular sorting of secretory proteins. Anterograde transport through the Golgi has been explained by the movement of Golgi cisternae, known as cisternal maturation. Because this explanation is now appreciated to be incomplete, interest has developed in understanding tubules that connect the Golgi cisternae. Here we show that the coat protein I (COPI) complex sorts anterograde cargoes into these tubules in human cells. Moreover, the small GTPase CDC42 regulates bidirectional Golgi transport by targeting the dual functions of COPI in cargo sorting and carrier formation. CDC42 also directly imparts membrane curvature to promote COPI tubule formation. Our findings further reveal that COPI tubular transport complements cisternal maturation in explaining how anterograde Golgi transport is achieved, and that bidirectional COPI transport is modulated by environmental cues through CDC42.

COPI acts in both vesicular and tubular transport.

Intracellular transport occurs through two general types of carrier, either vesicles or tubules. Coat proteins act as the core machinery that initiates vesicle formation, but the counterpart that initiates tubule formation has been unclear. Here, we find that the coat protein I (COPI) complex initially drives the formation of Golgi buds. Subsequently, a set of opposing lipid enzymatic activities determines whether these buds become vesicles or tubules. Lysophosphatidic acid acyltransferase-γ (LPAATγ) promotes COPI vesicle fission for retrograde vesicular transport. In contrast, cytosolic phospholipase A2-α (cPLA2α) inhibits this fission event to induce COPI tubules, which act in anterograde intra-Golgi transport and Golgi ribbon formation. These findings not only advance a molecular understanding of how COPI vesicle fission is achieved, but also provide insight into how COPI acts in intra-Golgi transport and reveal an unexpected mechanistic relationship between vesicular and tubular transport.

ARFGAP1 promotes AP-2-dependent endocytosis.

COPI (coat protein I) and the clathrin-AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport.

Mechanisms of COPI vesicle formation.

Coat Protein I (COPI) is one of the most intensely investigated coat complexes. Numerous studies have contributed to a general understanding of how coat proteins act to initiate intracellular vesicular transport. This review highlights key recent findings that have shaped our current understanding of how COPI vesicles are formed.

The evolving understanding of COPI vesicle formation.

The coat protein I (COPI) complex is considered to be one of the best-characterized coat complexes. Studies on how it functions in vesicle formation have provided seminal contributions to the general paradigm in vesicular transport that the ADP-ribosylation factor (ARF) small GTPases are key regulators of coat complexes. Here, we discuss emerging evidence that suggests the need to revise some long-held views on how COPI vesicle formation is achieved.

A role for the host coatomer and KDEL receptor in early vaccinia biogenesis.

Members of the poxvirus family have been investigated for their applications as vaccines and expression vectors and, more recently, because of concern for their potential as biological weapons. Vaccinia virus, the prototypic member, evolves through multiple forms during its replication. Here, we show a surprising way by which vaccinia hijacks coatomer for early viral biogenesis. Whereas coatomer forms COPI vesicles in the host early secretory system, vaccinia formation bypasses this role of coatomer, but instead, depends on coatomer interacting with the host KDEL receptor. To gain insight into the viral roles of these two host proteins, we have detected them on the earliest recognized viral forms. These findings not only suggest insights into early vaccinia biogenesis but also reveal an alternate mechanism by which coatomer acts.

A role for phosphatidic acid in COPI vesicle fission yields insights into Golgi maintenance.

Proteins essential for vesicle formation by the Coat Protein I (COPI) complex are being identified, but less is known about the role of specific lipids. Brefeldin-A ADP-ribosylated substrate (BARS) functions in the fission step of COPI vesicle formation. Here, we show that BARS induces membrane curvature in cooperation with phosphatidic acid. This finding has allowed us to further delineate COPI vesicle fission into two sub-stages: 1) an earlier stage of bud-neck constriction, in which BARS and other COPI components are required, and 2) a later stage of bud-neck scission, in which phosphatidic acid generated by phospholipase D2 (PLD2) is also required. Moreover, in contrast to the disruption of the Golgi seen on perturbing the core COPI components (such as coatomer), inhibition of PLD2 causes milder disruptions, suggesting that such COPI components have additional roles in maintaining Golgi structure other than through COPI vesicle formation.

Key components of the fission machinery are interchangeable.

Brefeldin-A ADP-ribosylated substrate (BARS) and dynamin function in membrane fission in distinct intracellular transport pathways, but whether their functions are mechanistically similar is unclear. Here, we show that ARFGAP1, a GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), couples to either BARS or endophilin B for vesicle formation by the coat protein I (COPI) complex - a finding that reveals an unanticipated mechanistic flexibility in mammalian COPI transport. Because dynamin is coupled to endophilin A in vesicle formation by the clathrin-coat complex, our finding also predicts that dynamin and ARF GAPs are likely to be functional counterparts in membrane fission among different transport pathways that connect intracellular membrane compartments.

A role for BARS at the fission step of COPI vesicle formation from Golgi membrane.

The core complex of Coat Protein I (COPI), known as coatomer, is sufficient to induce coated vesicular-like structures from liposomal membrane. In the context of biological Golgi membrane, both palmitoyl-coenzyme A (p-coA) and ARFGAP1, a GTPase-activating protein (GAP) for ADP-Ribosylation Factor 1, also participate in vesicle formation, but how their roles may be linked remains unknown. Moreover, whether COPI vesicle formation from Golgi membrane requires additional factors also remains unclear. We now show that Brefeldin-A ADP-Ribosylated Substrate (BARS) plays a critical role in the fission step of COPI vesicle formation from Golgi membrane. This role of BARS requires its interaction with ARFGAP1, which is in turn regulated oppositely by p-coA and nicotinamide adenine dinucleotide, which act as cofactors of BARS. Our findings not only identify a new factor needed for COPI vesicle formation from Golgi membrane but also reveal a surprising mechanism by which the roles of p-coA and GAP are linked in this process.

ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation.

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.