Uncovering mechanisms of interorganelle lipid transport by enzymatic mass tagging.

Lipid trafficking is critical for the biogenesis and expansion of organelle membranes. Lipid transport proteins (LTPs) have been proposed to facilitate lipid transport at contact sites between organelles. Despite the fundamental importance of LTPs in cell physiology, our knowledge on the mechanisms of interorganelle lipid distribution remains poor due to the scarcity of assays to monitor lipid flux in vivo. In this review, we highlight the recent development of a versatile method named METALIC (Mass tagging-Enabled Tracking of Lipids in Cells), which uses a combination of enzymatic mass tagging and mass spectrometry to track lipid flux between organelles inside living cells. We discuss the methodology, its distinct advantages, limitations as well as its potential to unearth the pipelines of lipid transport and LTP function in vivo.

METALIC reveals interorganelle lipid flux in live cells by enzymatic mass tagging.

The distinct activities of organelles depend on the proper function of their membranes. Coordinated membrane biogenesis of different organelles necessitates lipid transport from their site of synthesis to their destination. Several factors have been proposed to participate in lipid distribution, but despite its basic importance, in vivo evidence linking the absence of putative transport pathways to specific transport defects remains scarce. A reason for this scarcity is the near absence of in vivo lipid trafficking assays. Here we introduce a versatile method named METALIC (Mass tagging-Enabled TrAcking of Lipids In Cells) to track interorganelle lipid flux inside cells. In this strategy, two enzymes, one directed to a 'donor' and the other to an 'acceptor' organelle, add two distinct mass tags to lipids. Mass-spectrometry-based detection of lipids bearing the two mass tags is then used to quantify exchange between the two organelles. By applying this approach, we show that the ERMES and Vps13-Mcp1 complexes have transport activity in vivo, and unravel their relative contributions to endoplasmic reticulum-mitochondria lipid exchange.

Rewiring phospholipid biosynthesis reveals resilience to membrane perturbations and uncovers regulators of lipid homeostasis.

The organelles of eukaryotic cells differ in their membrane lipid composition. This heterogeneity is achieved by the localization of lipid synthesizing and modifying enzymes to specific compartments, as well as by intracellular lipid transport that utilizes vesicular and non-vesicular routes to ferry lipids from their place of synthesis to their destination. For instance, the major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), can be produced by multiple pathways and, in the case of PE, also at multiple locations. However, the molecular components that underlie lipid homeostasis as well as the routes allowing their distribution remain unclear. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. Among the candidates identified, we find genes linked to transcriptional regulation of lipid homeostasis, lipid metabolism, and transport. In particular, we identify a requirement for Csf1-an uncharacterized protein harboring a Chorein-N lipid transport motif-for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating Csf1 in interorganelle lipid transport and homeostatic adaptation.

Csf1: A Putative Lipid Transport Protein Required for Homeoviscous Adaptation of the Lipidome.

The non-vesicular transport of lipids between organelles mediated by lipid transport proteins (LTPs) is a key determinant of organelle biogenesis and function. Despite performing a vital function in organelle homeostasis, none of the LTP-encoding genes identified so far are truly essential, even in the simple genome of yeast, suggesting widespread redundancy. In line with this fact, it has been found that a number of LTPs have overlapping functions, making it challenging to assign unique roles for an individual LTP in lipid distribution. In our genetic screens under stringent conditions in which the distinct function of an LTP might become essential, we stumbled upon Csf1, a highly conserved protein with a Chorein-N motif found in other lipid transporters and unraveled a new function for Csf1 in lipid remodeling and homeoviscous adaptation of the lipidome. Here, we further speculate on the potential mechanisms of how the putative function of Csf1 in lipid transport could be intimately connected to its role in lipid remodeling across organelles.

Leri: A web-server for identifying protein functional networks from evolutionary couplings.

Information on the co-evolution of amino acid pairs in a protein can be used for endeavors such as protein engineering, mutation design, and structure prediction. Here we report a method that captures significant determinants of proteins using estimated co-evolution information to identify networks of residues, termed "residue communities", relevant to protein function. On the benchmark dataset (67 proteins with both catalytic and allosteric residues), the Pearson's correlation between the identified residues in the communities at functional sites is 0.53, and it is higher than 0.8 by taking account of conserved residues derived from the method. On the endoplasmic reticulum-mitochondria encounter structure complex, the results indicate three distinguishable residue communities that are relevant to functional roles in the protein family, suggesting that the residue communities could be general evolutionary signatures in proteins. Based on the method, we provide a webserver for the scientific community to explore the signatures in protein families, which establishes a powerful tool to analyze residue-level profiling for the discovery of functional sites and biological pathway identification. This web-server is freely available for non-commercial users at https://kornmann.bioch.ox.ac.uk/leri/services/ecs.html, neither login nor e-mail required.

Vps13-Mcp1 interact at vacuole-mitochondria interfaces and bypass ER-mitochondria contact sites.

Membrane contact sites between endoplasmic reticulum (ER) and mitochondria, mediated by the ER-mitochondria encounter structure (ERMES) complex, are critical for mitochondrial homeostasis and cell growth. Defects in ERMES can, however, be bypassed by point mutations in the endosomal protein Vps13 or by overexpression of the mitochondrial protein Mcp1. How this bypass operates remains unclear. Here we show that the mitochondrial outer membrane protein Mcp1 functions in the same pathway as Vps13 by recruiting it to mitochondria and promoting its association to vacuole-mitochondria contacts. Our findings support a model in which Mcp1 and Vps13 work as functional effectors of vacuole-mitochondria contact sites, while tethering is mediated by other factors, including Vps39. Tethered and functionally active vacuole-mitochondria interfaces then compensate for the loss of ERMES-mediated ER-mitochondria contact sites.

ER-mitochondrial junctions can be bypassed by dominant mutations in the endosomal protein Vps13.

The endoplasmic reticulum-mitochondria encounter structure (ERMES) complex tethers the endoplasmic reticulum and the mitochondria. It is thought to facilitate interorganelle lipid exchange and influence mitochondrial dynamics and mitochondrial DNA maintenance. Despite this important role, ERMES is not found in metazoans. Here, we identified single amino acid substitutions in Vps13 (vacuolar protein sorting 13), a large universally conserved eukaryotic protein, which suppress all measured phenotypic consequences of ERMES deficiency. Combined loss of VPS13 and ERMES is lethal, indicating that Vps13 and ERMES function in redundant pathways. Vps13 dynamically localizes to vacuole-mitochondria and to vacuole-nucleus contact sites depending on growth conditions, suggesting that ERMES function can be bypassed by the activity of other contact sites, and that contact sites establish a growth condition-regulated organelle network.

ER-mitochondria contact sites in yeast: beyond the myths of ERMES.

A standout feature of eukaryotic cells is the presence of organelles with distinct chemical compositions and physical properties, which aid in the accomplishment of specialized metabolic tasks. This complex topology, however, makes a permanent crosstalk between the organelles a necessity for the coordination of cellular function. While molecule exchange between organelles via the vesicular transport system has been extensively studied, communication via direct connections has only recently become a new matter of interest. These direct connections termed membrane contact sites (MCSs) represent zones of close proximity (10-30nm) between two organelles. Research in the past years has revealed a number of MCSs especially between the ER and almost every other organelle [1(•)]. In particular, the MCSs between the ER and the mitochondria have undergone intense investigation. While the quest for ER-mitochondria MCS components in human cells has led to the revelation of an ever growing number of potential factors, studies in the simpler eukaryote Saccharomyces cerevisiae revealed the actual existence of a molecular tether between the two organelles [2(••)].

The role of palmitoylation for protein recruitment to the inner membrane complex of the malaria parasite.

To survive and persist within its human host, the malaria parasite Plasmodium falciparum utilizes a battery of lineage-specific innovations to invade and multiply in human erythrocytes. With central roles in invasion and cytokinesis, the inner membrane complex, a Golgi-derived double membrane structure underlying the plasma membrane of the parasite, represents a unique and unifying structure characteristic to all organisms belonging to a large phylogenetic group called Alveolata. More than 30 structurally and phylogenetically distinct proteins are embedded in the IMC, where a portion of these proteins displays N-terminal acylation motifs. Although N-terminal myristoylation is catalyzed co-translationally within the cytoplasm of the parasite, palmitoylation takes place at membranes and is mediated by palmitoyl acyltransferases (PATs). Here, we identify a PAT (PfDHHC1) that is exclusively localized to the IMC. Systematic phylogenetic analysis of the alveolate PAT family reveals PfDHHC1 to be a member of a highly conserved, apicomplexan-specific clade of PATs. We show that during schizogony this enzyme has an identical distribution like two dual-acylated, IMC-localized proteins (PfISP1 and PfISP3). We used these proteins to probe into specific sequence requirements for IMC-specific membrane recruitment and their interaction with differentially localized PATs of the parasite.

The BLOC-1 complex promotes endosomal maturation by recruiting the Rab5 GTPase-activating protein Msb3.

Membrane microcompartments of the early endosomes serve as a sorting and signaling platform, where receptors are either recycled back to the plasma membrane or forwarded to the lysosome for destruction. In metazoan cells, three complexes, termed BLOC-1 to -3, mediate protein sorting from the early endosome to lysosomes and lysosome-related organelles. We now demonstrate that BLOC-1 is an endosomal Rab-GAP (GTPase-activating protein) adapter complex in yeast. The yeast BLOC-1 consisted of six subunits, which localized interdependently to the endosomes in a Rab5/Vps21-dependent manner. In the absence of BLOC-1 subunits, the balance between recycling and degradation of selected cargoes was impaired. Additionally, our data show that BLOC-1 is both a Vps21 effector and an adapter for its GAP Msb3. BLOC-1 and Msb3 interacted in vivo, and both mutants resulted in a redistribution of active Vps21 to the vacuole surface. We thus conclude that BLOC-1 controls the lifetime of active Rab5/Vps21 and thus endosomal maturation along the endocytic pathway.

Dissection of minimal sequence requirements for rhoptry membrane targeting in the malaria parasite.

Rhoptries are specialized secretory organelles characteristic of single cell organisms belonging to the clade Apicomplexa. These organelles play a key role in the invasion process of host cells by accumulating and subsequently secreting an unknown number of proteins mediating host cell entry. Despite their essential role, little is known about their biogenesis, components and targeting determinants. Here, we report on a conserved apicomplexan protein termed Armadillo Repeats-Only (ARO) protein that we localized to the cytosolic face of Plasmodium falciparum and Toxoplasma gondii rhoptries. We show that the first 20 N-terminal amino acids are sufficient for rhoptry membrane targeting. This protein relies on both - myristoylation and palmitoylation motifs - for membrane attachment. Although these lipid modifications are essential, they are not sufficient to direct ARO to the rhoptry membranes. Mutational analysis revealed additional residues within the first 20 amino acids of ARO that play an important role for rhoptry membrane attachment: the positively charged residues R9 and K14. Interestingly, the exchange of R9 with a negative charge entirely abolishes membrane attachment, whereas the exchange of K14 (and to a lesser extent K16) alters only its membrane specificity. Additionally, 17 proteins predicted to be myristoylated and palmitoylated in the first 20 N-terminal amino acids were identified in the genome of the malaria parasite. While most of the corresponding GFP fusion proteins were trafficked to the parasite plasma membrane, two were sorted to the apical organelles. Interestingly, these proteins have a similar motif identified for ARO.

Analysis of DHHC acyltransferases implies overlapping substrate specificity and a two-step reaction mechanism.

Asp-His-His-Cys (DHHC) cysteine-rich domain (CRD) acyltransferases are polytopic transmembrane proteins that are found along the endomembrane system of eukaryotic cells and mediate palmitoylation of peripheral and integral membrane proteins. Here, we address the in vivo substrate specificity of five of the seven DHHC acyltransferases for peripheral membrane proteins by an overexpression approach. For all analysed DHHC proteins we detect strongly overlapping substrate specificity. In addition, we now show acyltransferase activity for Pfa5. More importantly, the DHHC protein Pfa3 is able to trap several substrates at the vacuole. For Pfa3 and its substrate Vac8, we can distinguish two consecutive steps in the acylation reaction: an initial binding that occurs independently of its central cysteine in the DHHC box, but requires myristoylation of its substrate Vac8, and a DHHC-motif dependent acylation. Our data also suggest that proteins can be palmitoylated on several organelles. Thus, the intracellular distribution of DHHC proteins provides an acyltransferase network, which may promote dynamic membrane association of substrate proteins.