Motors, anchors, and connectors: orchestrators of organelle inheritance.

Organelle inheritance is a process whereby organelles are actively distributed between dividing cells at cytokinesis. Much valuable insight into the molecular mechanisms of organelle inheritance has come from the analysis of asymmetrically dividing cells, which transport a portion of their organelles to the bud while retaining another portion in the mother cell. Common principles apply to the inheritance of all organelles, although individual organelles use specific factors for their partitioning. Inheritance factors can be classified as motors, which are required for organelle transport; anchors, which immobilize organelles at distinct cell structures; or connectors, which mediate the attachment of organelles to motors and anchors. Here, we provide an overview of recent advances in the field of organelle inheritance and highlight how motor, anchor, and connector molecules choreograph the segregation of a multicopy organelle, the peroxisome. We also discuss the role of organelle population control in the generation of cellular diversity.

Peroxisome population control by phosphoinositide signaling at the endoplasmic reticulum-plasma membrane interface.

Phosphoinositides are lipid signaling molecules acting at the interface of membranes and the cytosol to regulate membrane trafficking, lipid transport and responses to extracellular stimuli. Peroxisomes are multicopy organelles that are highly responsive to changes in metabolic and environmental conditions. In yeast, peroxisomes are tethered to the cell cortex at defined focal structures containing the peroxisome inheritance protein, Inp1p. We investigated the potential impact of changes in cortical phosphoinositide levels on the peroxisome compartment of the yeast cell. Here we show that the phosphoinositide, phosphatidylinositol-4-phosphate (PI4P), found at the junction of the cortical endoplasmic reticulum and plasma membrane (cER-PM) acts to regulate the cell's peroxisome population. In cells lacking a cER-PM tether or the enzymatic activity of the lipid phosphatase Sac1p, cortical PI4P is elevated, peroxisome numbers and motility are increased, and peroxisomes are no longer firmly tethered to Inp1p-containing foci. Reattachment of the cER to the PM through an artificial ER-PM "staple" in cells lacking the cER-PM tether does not restore peroxisome populations to the wild-type condition, demonstrating that integrity of PI4P signaling at the cell cortex is required for peroxisome homeostasis.

Peroxisome-Mediated Metabolism Is Required for Immune Response to Microbial Infection.

The innate immune response is critical for animal homeostasis and is conserved from invertebrates to vertebrates. This response depends on specialized cells that recognize, internalize, and destroy microbial invaders through phagocytosis. This is coupled to autonomous or non-autonomous cellular signaling via reactive oxygen species (ROS) and cytokine production. Lipids are known signaling factors in this process, as the acute phase response of macrophages is accompanied by systemic lipid changes that help resolve inflammation. We found that peroxisomes, membrane-enclosed organelles central to lipid metabolism and ROS turnover, were necessary for the engulfment of bacteria by Drosophila and mouse macrophages. Peroxisomes were also required for resolution of bacterial infection through canonical innate immune signaling. Reduced peroxisome function impaired the turnover of the oxidative burst necessary to fight infection. This impaired response to bacterial challenge affected cell and organism survival and revealed a previously unknown requirement for peroxisomes in phagocytosis and innate immunity.

The Canadian Rare Diseases Models and Mechanisms (RDMM) Network: Connecting Understudied Genes to Model Organisms.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

Author Correction: Genome-wide SWAp-Tag yeast libraries for proteome exploration.

The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.

Molecular mechanisms of organelle inheritance: lessons from peroxisomes in yeast.

Preserving a functional set of cytoplasmic organelles in a eukaryotic cell requires a process of accurate organelle inheritance at cell division. Studies of peroxisome inheritance in yeast have revealed that polarized transport of a subset of peroxisomes to the emergent daughter cell is balanced by retention mechanisms operating in both mother cell and bud to achieve an equitable distribution of peroxisomes between them. It is becoming apparent that some common mechanistic principles apply to the inheritance of all organelles, but at the same time, inheritance factors specific for each organelle type allow the cell to differentially and specifically control the inheritance of its different organelle populations.

Pex20p functions as the receptor for non-PTS1/non-PTS2 acyl-CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica.

Most soluble proteins targeted to the peroxisomal matrix contain a C-terminal peroxisome targeting signal type 1 (PTS1) or an N-terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl-CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co-receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2-containing protein 3-ketoacyl-CoA thiolase (Pot1p) but also the non-PTS1/non-PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C-terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C-terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non-PTS1/non-PTS2 Aox isozymes into peroxisomes.

Determinants of the assembly, integrity and maintenance of the endoplasmic reticulum-peroxisome tether.

Organelle tethering and intercommunication are crucial for proper cell function. We previously described a tether between peroxisomes and the endoplasmic reticulum (ER) that acts in peroxisome population control in the yeast, Saccharomyces cerevisiae. Components of this tether are Pex3p, an integral membrane protein of both peroxisomes and the ER and Inp1p, a connector that links peroxisomes to the ER. Here, we report the analysis of random Inp1p mutants that enabled identification of regions in Inp1p required for the assembly and maintenance of the ER-peroxisome tether. Interaction analysis between Inp1p mutants and known Inp1p-binding proteins demonstrated that Pex3p and Inp1p do not constitute the sole components of the ER-peroxisome tether. Deletion of these Inp1p interactors whose steady-state localization is outside of ER-peroxisome tethers affected peroxisome dynamics. Our findings are consistent with the presence of regulatory cues that act on ER-peroxisome tethers and point to the existence of membrane contact sites between peroxisomes and organelles other than the ER.

Genome-wide SWAp-Tag yeast libraries for proteome exploration.

Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.

Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis.

Blastocystis is the most prevalent eukaryotic microbe colonizing the human gut, infecting approximately 1 billion individuals worldwide. Although Blastocystis has been linked to intestinal disorders, its pathogenicity remains controversial because most carriers are asymptomatic. Here, the genome sequence of Blastocystis subtype (ST) 1 is presented and compared to previously published sequences for ST4 and ST7. Despite a conserved core of genes, there is unexpected diversity between these STs in terms of their genome sizes, guanine-cytosine (GC) content, intron numbers, and gene content. ST1 has 6,544 protein-coding genes, which is several hundred more than reported for ST4 and ST7. The percentage of proteins unique to each ST ranges from 6.2% to 20.5%, greatly exceeding the differences observed within parasite genera. Orthologous proteins also display extreme divergence in amino acid sequence identity between STs (i.e., 59%-61% median identity), on par with observations of the most distantly related species pairs of parasite genera. The STs also display substantial variation in gene family distributions and sizes, especially for protein kinase and protease gene families, which could reflect differences in virulence. It remains to be seen to what extent these inter-ST differences persist at the intra-ST level. A full 26% of genes in ST1 have stop codons that are created on the mRNA level by a novel polyadenylation mechanism found only in Blastocystis. Reconstructions of pathways and organellar systems revealed that ST1 has a relatively complete membrane-trafficking system and a near-complete meiotic toolkit, possibly indicating a sexual cycle. Unlike some intestinal protistan parasites, Blastocystis ST1 has near-complete de novo pyrimidine, purine, and thiamine biosynthesis pathways and is unique amongst studied stramenopiles in being able to metabolize α-glucans rather than ß-glucans. It lacks all genes encoding heme-containing cytochrome P450 proteins. Predictions of the mitochondrion-related organelle (MRO) proteome reveal an expanded repertoire of functions, including lipid, cofactor, and vitamin biosynthesis, as well as proteins that may be involved in regulating mitochondrial morphology and MRO/endoplasmic reticulum (ER) interactions. In sharp contrast, genes for peroxisome-associated functions are absent, suggesting Blastocystis STs lack this organelle. Overall, this study provides an important window into the biology of Blastocystis, showcasing significant differences between STs that can guide future experimental investigations into differences in their virulence and clarifying the roles of these organisms in gut health and disease.

A Systematic Cell-Based Analysis of Localization of Predicted Drosophila Peroxisomal Proteins.

Peroxisomes are membrane-bound organelles found in almost all eukaryotic cells. They perform specialized biochemical functions that vary with organism, tissue or cell type. Mutations in human genes required for the assembly of peroxisomes result in a spectrum of diseases called the peroxisome biogenesis disorders. A previous sequence-based comparison of the predicted proteome of Drosophila melanogaster (the fruit fly) to human proteins identified 82 potential homologues of proteins involved in peroxisomal biogenesis, homeostasis or metabolism. However, the subcellular localization of these proteins relative to the peroxisome was not determined. Accordingly, we tested systematically the localization and selected functions of epitope-tagged proteins in Drosophila Schneider 2 cells to determine the subcellular localization of 82 potential Drosophila peroxisomal protein homologues. Excluding the Pex proteins, 34 proteins localized primarily to the peroxisome, 8 showed dual localization to the peroxisome and other structures, and 26 localized exclusively to organelles other than the peroxisome. Drosophila is a well-developed laboratory animal often used for discovery of gene pathways, including those linked to human disease. Our work establishes a basic understanding of peroxisome protein localization in Drosophila. This will facilitate use of Drosophila as a genetically tractable, multicellular model system for studying key aspects of human peroxisome disease.

Reconstitution of human peroxisomal ß-oxidation in yeast.

We report the permanent introduction of the human peroxisomal ß-oxidation enzymatic machinery required for straight chain degradation of fatty acids into the yeast, Saccharomyces cerevisiae. Peroxisomal ß-oxidation encompasses four sequential reactions that are confined to three enzymes. The genes encoding human acyl-CoA oxidase 1, peroxisomal multifunctional enzyme type 2 and 3-ketoacyl-CoA thiolase were introduced into the genomic loci of their yeast gene equivalents. The human ß-oxidation genes were individually tagged with sequence coding for GFP and expression of the protein chimeras as well as their targeting to peroxisomes was confirmed. Functional complementation of the ß-oxidation pathway was assessed by growth on media containing fatty acids of different chain lengths. Yeast cells exhibited distinctive substrate specificities depending on whether they expressed the human or their endogenous ß-oxidation machinery. The genetic engineering of yeast to contain a 'humanized' organelle is a first step for the in vivo study of human peroxisome disorders in a model organism.

Transport and retention mechanisms govern lipid droplet inheritance in Saccharomyces cerevisiae.

Lipid droplets are ubiquitous cellular structures involved in energy homeostasis and metabolism that have long been considered as simple inert deposits of lipid. Here, we show that lipid droplets are bona fide organelles that are actively partitioned between mother cell and daughter cell in Saccharomyces cerevisiae. Video microscopy revealed that a subset of lipid droplets moves from mother cell to bud in an ordered, vectorial process, while the remaining lipid droplets are retained by the mother cell. Bud-directed movement of lipid droplets is mediated by the molecular motor Myo2p, while retention of lipid droplets occurs at the perinuclear endoplasmic reticulum. Lipid droplets are thus apportioned between mother cell and daughter cell at cell division rather than being made anew.

Peroxins Pex30 and Pex29 Dynamically Associate with Reticulons to Regulate Peroxisome Biogenesis from the Endoplasmic Reticulum.

Peroxisome proliferation occurs by at least two routes, division of existing peroxisomes and de novo biogenesis from the endoplasmic reticulum (ER). The proteins and molecular mechanisms governing peroxisome emergence from the ER are poorly characterized. In this study, we report that two integral membrane peroxins (proteins required for peroxisome biogenesis) in Saccharomyces cerevisiae, Pex29 and Pex30, reside in distinct regions of the ER and associate with Rtn1 and Yop1, reticulon family members that contribute to ER morphology, to govern peroxisome emergence from the ER. In vivo and in vitro analyses reveal that peroxisome proliferation is therefore not restricted to the peroxisome but begins at the level of the ER.

Sharing with your children: Mechanisms of peroxisome inheritance.

Organelle inheritance is the process by which eukaryotic cells actively replicate and equitably partition their organelles between mother cell and daughter cell at cytokinesis to maintain the benefits of subcellular compartmentalization. The budding yeast Saccharomyces cerevisiae has proven invaluable in helping to define the factors involved in the inheritance of different organelles and in understanding how these factors act and interact to maintain balance in the organelle populations of actively dividing cells. Inheritance factors can be classified as motors that transport organelles, tethers that retain organelles, and connectors (receptors) that mediate the attachment of organelles to motors and anchors. This article will review how peroxisomes are inherited by cells, with a focus on budding yeast, and will discuss common themes and mechanisms of action that underlie the inheritance of all membrane-enclosed organelles.

An ER-peroxisome tether exerts peroxisome population control in yeast.

Eukaryotic cells compartmentalize biochemical reactions into membrane-enclosed organelles that must be faithfully propagated from one cell generation to the next. Transport and retention processes balance the partitioning of organelles between mother and daughter cells. Here we report the identification of an ER-peroxisome tether that links peroxisomes to the ER and ensures peroxisome population control in the yeast Saccharomyces cerevisiae. The tether consists of the peroxisome biogenic protein, Pex3p, and the peroxisome inheritance factor, Inp1p. Inp1p bridges the two compartments by acting as a molecular hinge between ER-bound Pex3p and peroxisomal Pex3p. Asymmetric peroxisome division leads to the formation of Inp1p-containing anchored peroxisomes and Inp1p-deficient mobile peroxisomes that segregate to the bud. While peroxisomes in mother cells are not released from tethering, de novo formation of tethers in the bud assists in the directionality of peroxisome transfer. Peroxisomes are thus stably maintained over generations of cells through their continued interaction with tethers.

Sharing the cell's bounty - organelle inheritance in yeast.

Eukaryotic cells replicate and partition their organelles between the mother cell and the daughter cell at cytokinesis. Polarized cells, notably the budding yeast Saccharomyces cerevisiae, are well suited for the study of organelle inheritance, as they facilitate an experimental dissection of organelle transport and retention processes. Much progress has been made in defining the molecular players involved in organelle partitioning in yeast. Each organelle uses a distinct set of factors - motor, anchor and adaptor proteins - that ensures its inheritance by future generations of cells. We propose that all organelles, regardless of origin or copy number, are partitioned by the same fundamental mechanism involving division and segregation. Thus, the mother cell keeps, and the daughter cell receives, their fair and equitable share of organelles. This mechanism of partitioning moreover facilitates the segregation of organelle fragments that are not functionally equivalent. In this Commentary, we describe how this principle of organelle population control affects peroxisomes and other organelles, and outline its implications for yeast life span and rejuvenation.

An ancestral role in peroxisome assembly is retained by the divisional peroxin Pex11 in the yeast Yarrowia lipolytica.

The peroxin Pex11 has a recognized role in peroxisome division. Pex11p remodels and elongates peroxisomal membranes prior to the recruitment of dynamin-related GTPases that act in membrane scission to divide peroxisomes. We performed a comprehensive comparative genomics survey to understand the significance of the evolution of the Pex11 protein family in yeast and other eukaryotes. Pex11p is highly conserved and ancestral, and has undergone numerous lineage-specific duplications, whereas other Pex11 protein family members are fungal-specific innovations. Functional characterization of the in-silico-predicted Pex11 protein family members of the yeast Yarrowia lipolytica, i.e. Pex11p, Pex11Cp and Pex11/25p, demonstrated that Pex11Cp and Pex11/25p have a role in the regulation of peroxisome size and number characteristic of Pex11 protein family members. Unexpectedly, deletion of PEX11 in Y. lipolytica produces cells that lack morphologically identifiable peroxisomes, mislocalize peroxisomal matrix proteins and preferentially degrade peroxisomal membrane proteins, i.e. they exhibit the classical pex mutant phenotype, which has not been observed previously in cells deleted for the PEX11 gene. Our results are consistent with an unprecedented role for Pex11p in de novo peroxisome assembly.

Flavivirus Infection Impairs Peroxisome Biogenesis and Early Antiviral Signaling.

UNLABELLED: Flaviviruses are significant human pathogens that have an enormous impact on the global health burden. Currently, there are very few vaccines against or therapeutic treatments for flaviviruses, and our understanding of how these viruses cause disease is limited. Evidence suggests that the capsid proteins of flaviviruses play critical nonstructural roles during infection, and therefore, elucidating how these viral proteins affect cellular signaling pathways could lead to novel targets for antiviral therapy. We used affinity purification to identify host cell proteins that interact with the capsid proteins of West Nile and dengue viruses. One of the cellular proteins that formed a stable complex with flavivirus capsid proteins is the peroxisome biogenesis factor Pex19. Intriguingly, flavivirus infection resulted in a significant loss of peroxisomes, an effect that may be due in part to capsid expression. We posited that capsid protein-mediated sequestration and/or degradation of Pex19 results in loss of peroxisomes, a situation that could result in reduced early antiviral signaling. In support of this hypothesis, we observed that induction of the lambda interferon mRNA in response to a viral RNA mimic was reduced by more than 80%. Together, our findings indicate that inhibition of peroxisome biogenesis may be a novel mechanism by which flaviviruses evade the innate immune system during early stages of infection. IMPORTANCE: RNA viruses infect hundreds of millions of people each year, causing significant morbidity and mortality. Chief among these pathogens are the flaviviruses, which include dengue virus and West Nile virus. Despite their medical importance, there are very few prophylactic or therapeutic treatments for these viruses. Moreover, the manner in which they subvert the innate immune response in order to establish infection in mammalian cells is not well understood. Recently, peroxisomes were reported to function in early antiviral signaling, but very little is known regarding if or how pathogenic viruses affect these organelles. We report for the first time that flavivirus infection results in significant loss of peroxisomes in mammalian cells, which may indicate that targeting of peroxisomes is a key strategy used by viruses to subvert early antiviral defenses.