Structural insight into an Arl1-ArfGEF complex involved in Golgi recruitment of a GRIP-domain golgin.

Arl1 is an Arf-like (Arl) GTP-binding protein that interacts with the guanine nucleotide exchange factor Gea2 to recruit the golgin Imh1 to the Golgi. The Arl1-Gea2 complex also binds and activates the phosphatidylserine flippase Drs2 and these functions may be related, although the underlying molecular mechanism is unclear. Here we report high-resolution cryo-EM structures of the full-length Gea2 and the Arl1-Gea2 complex. Gea2 is a large protein with 1459 residues and is composed of six domains (DCB, HUS, SEC7, HDS1-3). We show that Gea2 assembles a stable dimer via an extensive interface involving hydrophobic and electrostatic interactions in the DCB and HUS region. Contrary to the previous report on a Gea2 homolog in which Arl1 binds to the dimerization surface of the DCB domain, implying a disrupted dimer upon Arl1 binding, we find that Arl1 binds to the outside surface of the Gea2 DCB domain, leaving the Gea2 dimer intact. The interaction between Arl1 and Gea2 involves the classic FWY aromatic residue triad as well as two Arl1-specific residues. We show that key mutations that disrupt the Arl1-Gea2 interaction abrogate Imh1 Golgi association. This work clarifies the Arl1-Gea2 interaction and improves our understanding of molecular events in the membrane trafficking.

Flipping the script: Advances in understanding how and why P4-ATPases flip lipid across membranes.

Type IV P-type ATPases (P4-ATPases) are a family of transmembrane enzymes that translocate lipid substrates from the outer to the inner leaflet of biological membranes and thus create an asymmetrical distribution of lipids within membranes. On the cellular level, this asymmetry is essential for maintaining the integrity and functionality of biological membranes, creating platforms for signaling events and facilitating vesicular trafficking. On the organismal level, this asymmetry has been shown to be important in maintaining blood homeostasis, liver metabolism, neural development, and the immune response. Indeed, dysregulation of P4-ATPases has been linked to several diseases; including anemia, cholestasis, neurological disease, and several cancers. This review will discuss the evolutionary transition of P4-ATPases from cation pumps to lipid flippases, the new lipid substrates that have been discovered, the significant advances that have been achieved in recent years regarding the structural mechanisms underlying the recognition and flipping of specific lipids across biological membranes, and the consequences of P4-ATPase dysfunction on cellular and physiological functions. Additionally, we emphasize the requirement for additional research to comprehensively understand the involvement of flippases in cellular physiology and disease and to explore their potential as targets for therapeutics in treating a variety of illnesses. The discussion in this review will primarily focus on the budding yeast, C. elegans, and mammalian P4-ATPases.

ATP10A deficiency results in male-specific infertility in mice.

Over 8% of couples worldwide are affected by infertility and nearly half of these cases are due to male-specific issues where the underlying cause is often unknown. Therefore, discovery of new genetic factors contributing to male-specific infertility in model organisms can enhance our understanding of the etiology of this disorder. Here we show that murine ATP10A, a phospholipid flippase, is highly expressed in male reproductive organs, specifically the testes and vas deferens. Therefore, we tested the influence of ATP10A on reproduction by examining fertility of Atp10A knockout mice. Our findings reveal that Atp10A deficiency leads to male-specific infertility, but does not perturb fertility in the females. The Atp10A deficient male mice exhibit smaller testes, reduced sperm count (oligozoospermia) and lower sperm motility (asthenozoospermia). Additionally, Atp10A deficient mice display testes and vas deferens histopathological abnormalities, as well as altered total and relative amounts of hormones associated with the hypothalamic-pituitary-gonadal axis. Surprisingly, circulating testosterone is elevated 2-fold in the Atp10A knockout mice while luteinizing hormone, follicle stimulating hormone, and inhibin B levels were not significantly different from WT littermates. The knockout mice also exhibit elevated levels of gonadotropin receptors and alterations to ERK, p38 MAPK, Akt, and cPLA2-dependent signaling in the testes. Atp10A was knocked out in the C57BL/6J background, which also carries an inactivating nonsense mutation in the closely related lipid flippase, Atp10D. We have corrected the Atp10D nonsense mutation using CRISPR/Cas9 and determined that loss of Atp10A alone is sufficient to cause infertility in male mice. Collectively, these findings highlight the critical role of ATP10A in male fertility in mice and provide valuable insights into the underlying molecular mechanisms.

Deficiency of the lipid flippase ATP10A causes diet-induced dyslipidemia in female mice.

Genetic association studies have linked ATP10A and closely related type IV P-type ATPases (P4-ATPases) to insulin resistance and vascular complications, such as atherosclerosis. ATP10A translocates phosphatidylcholine and glucosylceramide across cell membranes, and these lipids or their metabolites play important roles in signal transduction pathways regulating metabolism. However, the influence of ATP10A on lipid metabolism in mice has not been explored. Here, we generated gene-specific Atp10A knockout mice and show that Atp10A-/- mice fed a high-fat diet did not gain excess weight relative to wild-type littermates. However, Atp10A-/- mice displayed female-specific dyslipidemia characterized by elevated plasma triglycerides, free fatty acids and cholesterol, as well as altered VLDL and HDL properties. We also observed increased circulating levels of several sphingolipid species along with reduced levels of eicosanoids and bile acids. The Atp10A-/- mice also displayed hepatic insulin resistance without perturbations to whole-body glucose homeostasis. Thus, ATP10A has a sex-specific role in regulating plasma lipid composition and maintaining hepatic liver insulin sensitivity in mice.

Type IV P-Type ATPases: Recent Updates in Cancer Development, Progression, and Treatment.

Adaptations of cancer cells for survival are remarkable. One of the most significant properties of cancer cells to prevent the immune system response and resist chemotherapy is the altered lipid metabolism and resulting irregular cell membrane composition. The phospholipid distribution in the plasma membrane of normal animal cells is distinctly asymmetric. Lipid flippases are a family of enzymes regulating membrane asymmetry, and the main class of flippases are type IV P-type ATPases (P4-ATPases). Alteration in the function of flippases results in changes to membrane organization. For some lipids, such as phosphatidylserine, the changes are so drastic that they are considered cancer biomarkers. This review will analyze and discuss recent publications highlighting the role that P4-ATPases play in the development and progression of various cancer types, as well as prospects of targeting P4-ATPases for anti-cancer treatment.

Deficiency of the lipid flippase ATP10A causes diet-induced dyslipidemia in female mice.

Genetic association studies have linked ATP10A and closely related type IV P-type ATPases (P4-ATPases) to insulin resistance and vascular complications, such as atherosclerosis. ATP10A translocates phosphatidylcholine and glucosylceramide across cell membranes, and these lipids or their metabolites play important roles in signal transduction pathways regulating metabolism. However, the influence of ATP10A on lipid metabolism in mice has not been explored. Here, we generated gene-specific Atp10A knockout mice and show that Atp10A-/- mice fed a high-fat diet did not gain excess weight relative to wild-type littermates. However, Atp10A-/- mice displayed female-specific dyslipidemia characterized by elevated plasma triglycerides, free fatty acids and cholesterol, as well as altered VLDL and HDL properties. We also observed increased circulating levels of several sphingolipid species along with reduced levels of eicosanoids and bile acids. The Atp10A-/- mice also displayed hepatic insulin resistance without perturbations to whole-body glucose homeostasis. Thus, ATP10A has a sex-specific role in regulating plasma lipid composition and maintaining hepatic liver insulin sensitivity in mice.

The P4-ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast.

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.

An interaction between ß'-COP and the ArfGAP, Glo3, maintains post-Golgi cargo recycling.

The essential COPI coat mediates retrieval of transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAPs remain elusive. Biochemical and biophysical data reveal how ß'-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity. Calorimetry data demonstrate that both ß'-COP propeller domains are required to bind Glo3. An acidic patch on ß'-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (binding of coatomer, cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or ß'-COP abrogate the interaction in vitro, and loss of the ß'-COP/Glo3 interaction drives Ste2 missorting to the vacuole and aberrant Golgi morphology in budding yeast. These data suggest that cells require the ß'-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where ß'-COP serves as a molecular platform to coordinate binding to multiple proteins, including Glo3, Arf1, and the COPI F-subcomplex.

Quantification of Golgi Protein Mislocalization to the Budding Yeast Vacuole.

The localization of proteins to the Golgi complex is a dynamic process requiring sorting signals in the cytosolic domains of resident Golgi proteins and retrograde vesicular trafficking. Disruptions in these signals or in the retrograde pathways often lead to mislocalization of Golgi proteins to the vacuole in budding yeast. The extent of vacuolar mislocalization can be quantified through colocalization of GFP-tagged Golgi proteins with fluorescent dyes that mark either the vacuole limiting membrane or the vacuole lumen. Manders' colocalization coefficient (MCC) is a useful tool for quantifying the degree of colocalization. However, the dilution of fluorescence signal intensity that occurs when GFP-tagged Golgi proteins mislocalize to the much larger vacuole is problematic for thresholding the images prior to calculating the MCC. In this chapter, we describe the use of Multi-Otsu thresholding in ImageJ to quantify the degree of GFP-tagged protein mislocalization to the vacuole. Furthermore, these methods can be applied to other colocalization events within the cell.

Lipid Transport by Candida albicans Dnf2 Is Required for Hyphal Growth and Virulence.

Candida albicans is a common cause of human mucosal yeast infections, and invasive candidiasis can be fatal. Antifungal medications are limited, but those targeting the pathogen cell wall or plasma membrane have been effective. Therefore, virulence factors controlling membrane biogenesis are potential targets for drug development. P4-ATPases contribute to membrane biogenesis by selecting and transporting specific lipids from the extracellular leaflet to the cytoplasmic leaflet of the bilayer to generate lipid asymmetry. A subset of heterodimeric P4-ATPases, including Dnf1-Lem3 and Dnf2-Lem3 from Saccharomyces cerevisiae, transport phosphatidylcholine (PC), phosphatidylethanolamine (PE), and the sphingolipid glucosylceramide (GlcCer). GlcCer is a critical lipid for Candida albicans polarized growth and virulence, but the role of GlcCer transporters in virulence has not been explored. Here, we show that the Candida albicans Dnf2 (CaDnf2) requires association with CaLem3 to form a functional transporter and flip fluorescent derivatives of GlcCer, PC, and PE across the plasma membrane. Mutation of conserved substrate-selective residues in the membrane domain strongly abrogates GlcCer transport and partially disrupts PC transport by CaDnf2. Candida strains harboring dnf2-null alleles (dnf2ΔΔ) or point mutations that disrupt substrate recognition exhibit defects in yeast-to-hypha growth transition, filamentous growth, and virulence in systemically infected mice. The influence of CaDNF1 deletion on the morphological phenotypes is negligible, although the dnf1ΔΔ dnf2ΔΔ strain was less virulent than the dnf2ΔΔ strain. These results indicate that the transport of GlcCer and/or PC by plasma membrane P4-ATPases is important for the pathogenicity of Candida albicans.

Ubiquitination drives COPI priming and Golgi SNARE localization.

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime coatomer protein I (COPI) assembly to ensure incorporation of these essential cargoes into vesicles, but the regulation of these events is poorly understood. Here, we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs in Saccharomyces cerevisiae. The ability of COPI to bind ubiquitin, but not the dilysine motif, through its N-terminal WD repeat domain of ß'-COP or through an unrelated ubiquitin-binding domain is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3, and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is proposed to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.

An orthologous gene coevolution network provides insight into eukaryotic cellular and genomic structure and function.

The evolutionary rates of functionally related genes often covary. We present a gene coevolution network inferred from examining nearly 3 million orthologous gene pairs from 332 budding yeast species spanning ~400 million years of evolution. Network modules provide insight into cellular and genomic structure and function. Examination of the phenotypic impact of network perturbation using deletion mutant data from the baker's yeast Saccharomyces cerevisiae, which were obtained from previously published studies, suggests that fitness in diverse environments is affected by orthologous gene neighborhood and connectivity. Mapping the network onto the chromosomes of S. cerevisiae and Candida albicans revealed that coevolving orthologous genes are not physically clustered in either species; rather, they are often located on different chromosomes or far apart on the same chromosome. The coevolution network captures the hierarchy of cellular structure and function, provides a roadmap for genotype-to-phenotype discovery, and portrays the genome as a linked ensemble of genes.

AP-3 shows off its flexibility for the cryo-EM camera.

The tetrameric adaptor protein AP-3 is critical for the transport of proteins to lysosomes and lysosome-related organelles. The structures of homologous adaptors AP-1 and AP-2 have revealed a closed-to-open conformational change upon membrane recruitment and phosphoinositide binding. Recently, Schoppe et al. reported the first cryo-EM structures of AP-3 from budding yeast and described remarkably flexible solution structures that are all in the open conformation. The apparent lack of a closed conformational state, the first such description in the literature, allows AP-3 to be more reliant on cargo interaction for its initial membrane recruitment compared with AP-1.

Structural basis of the P4B ATPase lipid flippase activity.

P4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic α-subunit and accessory ß-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a ß-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1's ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.

Transport mechanism of P4 ATPase phosphatidylcholine flippases.

The P4 ATPases use ATP hydrolysis to transport large lipid substrates across lipid bilayers. The structures of the endosome- and Golgi-localized phosphatidylserine flippases-such as the yeast Drs2 and human ATP8A1-have recently been reported. However, a substrate-binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases with other substrates are unknown. Here, we report structures of the S. cerevisiae Dnf1-Lem3 and Dnf2-Lem3 complexes. We captured substrate phosphatidylcholine molecules on both the exoplasmic and cytosolic sides and found that they have similar structures. Unexpectedly, Lem3 contributes to substrate binding. The conformational transitions of these phosphatidylcholine transporters match those of the phosphatidylserine transporters, suggesting a conserved mechanism among P4 ATPases. Dnf1/Dnf2 have a unique P domain helix-turn-helix insertion that is important for function. Therefore, P4 ATPases may have retained an overall transport mechanism while evolving distinct features for different lipid substrates.

Exofacial membrane composition and lipid metabolism regulates plasma membrane P4-ATPase substrate specificity.

The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. Regulation of membrane asymmetry is a fundamental characteristic of membrane biology and is crucial for signal transduction, vesicle transport, and cell division. The type IV family of P-ATPases, or P4-ATPases, establishes membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is unclear how P4-ATPases sort through the spectrum of membrane lipids to identify their desired substrate(s) and how the membrane environment modulates this activity. Therefore, we tested how the yeast plasma membrane P4-ATPase, Dnf2, responds to changes in membrane composition induced by perturbation of endogenous lipid biosynthetic pathways or exogenous application of lipid. The primary substrates of Dnf2 are glucosylceramide (GlcCer) and phosphatidylcholine (PC, or their lyso-lipid derivatives), and we find that these substrates compete with each other for transport. Acutely inhibiting sphingolipid synthesis using myriocin attenuates transport of exogenously applied GlcCer without perturbing PC transport. Deletion of genes controlling later steps of glycosphingolipid production also perturb GlcCer transport to a greater extent than PC transport. In contrast, perturbation of ergosterol biosynthesis reduces PC and GlcCer transport equivalently. Surprisingly, application of lipids that are poor transport substrates differentially affects PC and GlcCer transport by Dnf2, thus altering substrate preference. Our data indicate that Dnf2 exhibits exquisite sensitivity to the membrane composition, thus providing feedback onto the function of the P4-ATPases.

Yeast synaptobrevin, Snc1, engages distinct routes of postendocytic recycling mediated by a sorting nexin, Rcy1-COPI, and retromer.

The budding yeast v-SNARE, Snc1, mediates fusion of exocytic vesicles to the plasma membrane (PM) and is subsequently recycled back to the Golgi. Postendocytic recycling of Snc1 requires a phospholipid flippase (Drs2-Cdc50), an F-box protein (Rcy1), a sorting nexin (Snx4-Atg20), and the COPI coat complex. A portion of the endocytic tracer FM4-64 is also recycled back to the PM after internalization. However, the relationship between Snx4, Drs2, Rcy1, and COPI in recycling Snc1 or FM4-64 is unclear. Here we show that rcy1∆ and drs2∆ single mutants, or a COPI mutant deficient in ubiquitin binding, display a defect in recycling FM4-64 while snx4∆ cells recycle FM4-64 normally. The addition of latrunculin A to acutely inhibit endocytosis shows that rcy1∆ and snx4∆ single mutants retain the ability to recycle Snc1, but a snx4∆rcy1∆ mutant substantially blocks export. Additional deletion of a retromer subunit completely eliminates recycling of Snc1 in the triple mutant (snx4∆rcy1∆vps35∆). A minor role for retromer in Snc1 recycling can also be observed in single and double mutants harboring vps35∆. These data support the existence of three distinct and parallel recycling pathways mediated by Drs2/Rcy1/COPI, Snx4-Atg20, and retromer that retrieve an exocytic v-SNARE from the endocytic pathway to the Golgi.

Mammalian Retromer Is an Adaptable Scaffold for Cargo Sorting from Endosomes.

Metazoan retromer (VPS26/VPS35/VPS29) associates with sorting nexins on endosomal tubules to sort proteins to the trans-Golgi network or plasma membrane. Mechanisms of metazoan retromer assembly remain undefined. We combine single-particle cryoelectron microscopy with biophysical methods to uncover multiple oligomer structures. 2D class averages reveal mammalian heterotrimers; dimers of trimers; tetramers of trimers; and flat chains. These species are further supported by biophysical solution studies. We provide reconstructions of all species, including key sub-structures (∼5 Å resolution). Local resolution variation suggests that heterotrimers and dimers adopt multiple conformations. Our structures identify a flexible, highly conserved electrostatic dimeric interface formed by VPS35 subunits. We generate structure-based mutants to disrupt this interface in vitro. Equivalent mutations in yeast demonstrate a mild cargo-sorting defect. Our data suggest the metazoan retromer is an adaptable and plastic scaffold that accommodates interactions with different sorting nexins to sort multiple cargoes from endosomes their final destinations.

Conserved mechanism of phospholipid substrate recognition by the P4-ATPase Neo1 from Saccharomyces cerevisiae.

The type IV P-type ATPases (P4-ATPases) thus far characterized are lipid flippases that transport specific substrates, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the exofacial leaflet to the cytofacial leaflet of membranes. This transport activity generates compositional asymmetry between the two leaflets important for signal transduction, cytokinesis, vesicular transport, and host-pathogen interactions. Most P4-ATPases function as a heterodimer with a ß-subunit from the Cdc50 protein family, but Neo1 from Saccharomyces cerevisiae and its metazoan orthologs lack a ß-subunit requirement and it is unclear how these proteins transport substrate. Here we tested if residues linked to lipid substrate recognition in other P4-ATPases also contribute to Neo1 function in budding yeast. Point mutations altering entry gate residues in the first (Q209A) and fourth (S457Q) transmembrane segments of Neo1, where phospholipid substrate would initially be selected, disrupt PS and PE membrane asymmetry, but do not perturb growth of cells. Mutation of both entry gate residues inactivates Neo1, and cells expressing this variant are inviable. We also identified a gain-of-function mutation in the second transmembrane segment of Neo1 (Neo1[Y222S]), predicted to help form the entry gate, that substantially enhances Neo1's ability to replace the function of a well characterized phospholipid flippase, Drs2, in establishing PS and PE asymmetry. These results suggest a common mechanism for substrate recognition in widely divergent P4-ATPases.