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.

Peroxisomes exhibit compromised structure and matrix protein content in SARS-CoV-2-infected cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has triggered global health and economic crises. Here we report the effects of SARS-CoV-2 infection on peroxisomes of human cell lines Huh-7 and SK-N-SH. Peroxisomes undergo dramatic changes in morphology in SARS-CoV-2-infected cells. Rearrangement of peroxisomal membranes is followed by redistribution of peroxisomal matrix proteins to the cytosol, resulting in a dramatic decrease in the number of mature peroxisomes. The SARS-CoV-2 ORF14 protein was shown to interact physically with human PEX14, a peroxisomal membrane protein required for matrix protein import and peroxisome biogenesis. Given the important roles of peroxisomes in innate immunity, SARS-CoV-2 may directly target peroxisomes, resulting in loss of peroxisome structural integrity, matrix protein content and ability to function in antiviral signaling.

The early-acting glycosome biogenic protein Pex3 is essential for trypanosome viability.

Trypanosomatid parasites are infectious agents for diseases such as African sleeping sickness, Chagas disease, and leishmaniasis that threaten millions of people, mostly in the emerging world. Trypanosomes compartmentalize glycolytic enzymes to an organelle called the glycosome, a specialized peroxisome. Functionally intact glycosomes are essential for trypanosomatid viability, making glycosomal proteins as potential drug targets against trypanosomatid diseases. Peroxins (Pex), of which Pex3 is the master regulator, control glycosome biogenesis. Although Pex3 has been found throughout the eukaryota, its identity has remained stubbornly elusive in trypanosomes. We used bioinformatics predictive of protein secondary structure to identify trypanosomal Pex3. Microscopic and biochemical analyses showed trypanosomal Pex3 to be glycosomal. Interaction of Pex3 with the peroxisomal membrane protein receptor Pex19 observed for other eukaryotes is replicated by trypanosomal Pex3 and Pex19. Depletion of Pex3 leads to mislocalization of glycosomal proteins to the cytosol, reduced glycosome numbers, and trypanosomatid death. Our findings are consistent with Pex3 being an essential gene in trypanosomes.

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.

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.

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.

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.

How peroxisomes partition between cells. A story of yeast, mammals and filamentous fungi.

Eukaryotic cells are subcompartmentalized into discrete, membrane-enclosed organelles. These organelles must be preserved in cells over many generations to maintain the selective advantages afforded by compartmentalization. Cells use complex molecular mechanisms of organelle inheritance to achieve high accuracy in the sharing of organelles between daughter cells. Here we focus on how a multi-copy organelle, the peroxisome, is partitioned in yeast, mammalian cells, and filamentous fungi, which differ in their mode of cell division. Cells achieve equidistribution of their peroxisomes through organelle transport and retention processes that act coordinately, although the strategies employed vary considerably by organism. Nevertheless, we propose that mechanisms common across species apply to the partitioning of all membrane-enclosed organelles.

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.

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.

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.

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.

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.

Doing the math: How yeast cells maintain their peroxisome populations.

The formation of membrane contact sites between cellular organelles is required for proper organelle communication and maintenance in the compartmentalized eukaryotic cell. We recently identified a tether that links peroxisomes to the cortical ER in the yeast, Saccharomyces cerevisiae. The tether is made up of the peroxisome biogenic protein Pex3p and the peroxisome inheritance factor Inp1p, and is formed by Inp1p-mediated linkage of ER-bound Pex3p and peroxisomal Pex3p. Here we discuss how this tether is fine-tuned to ensure that peroxisomes are stably maintained over generations of yeast cells.

Peroxisome biogenesis: something old, something new, something borrowed.

Eukaryotic cells are characterized by their varied complement of organelles. One set of membrane-bound, usually spherical compartments are commonly grouped together under the term peroxisomes. Peroxisomes function in regulating the synthesis and availability of many diverse lipids by harnessing the power of oxidative reactions and contribute to a number of metabolic processes essential for cellular differentiation and organismal development.

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.

Phosphorylation-dependent activation of peroxisome proliferator protein PEX11 controls peroxisome abundance.

Peroxisomes are dynamic organelles that divide continuously in growing cell cultures and expand extensively in lipid-rich medium. Peroxisome population control is achieved in part by Pex11p-dependent regulation of peroxisome size and number. Although the production of Pex11p in yeast is tightly linked to peroxisome biogenesis by transcriptional regulation of the PEX11 gene, it remains unclear if and how Pex11p activity could be modulated by rapid signaling. We report the reversible phosphorylation of Saccharomyces cerevisiae Pex11p in response to nutritional cues and delineate a mechanism for phosphorylation-dependent activation of Pex11p through the analysis of phosphomimicking mutants. Peroxisomal phenotypes in the PEX11-A and PEX11-D strains expressing constitutively dephosphorylated and phosphorylated forms of Pex11p resemble those of PEX11 gene knock-out and overexpression mutants, although PEX11 transcript and Pex11 protein levels remain unchanged. We demonstrate functional inequality and differences in subcellular localization of the Pex11p forms. Pex11Dp promotes peroxisome fragmentation when reexpressed in cells containing induced peroxisomes. Pex11p translocates between endoplasmic reticulum and peroxisomes in a phosphorylation-dependent manner, whereas Pex11Ap and Pex11Dp are impaired in trafficking and constitutively associated with mature and proliferating peroxisomes, respectively. Overexpression of cyclin-dependent kinase Pho85p results in hyperphosphorylation of Pex11p and peroxisome proliferation. This study provides the first evidence for control of peroxisome dynamics by phosphorylation-dependent regulation of a peroxin.

Myosin-driven peroxisome partitioning in S. cerevisiae.

In Saccharomyces cerevisiae, the class V myosin motor Myo2p propels the movement of most organelles. We recently identified Inp2p as the peroxisome-specific receptor for Myo2p. In this study, we delineate the region of Myo2p devoted to binding peroxisomes. Using mutants of Myo2p specifically impaired in peroxisome binding, we dissect cell cycle-dependent and peroxisome partitioning-dependent mechanisms of Inp2p regulation. We find that although total Inp2p levels oscillate with the cell cycle, Inp2p levels on individual peroxisomes are controlled by peroxisome inheritance, as Inp2p aberrantly accumulates and decorates all peroxisomes in mother cells when peroxisome partitioning is abolished. We also find that Inp2p is a phosphoprotein whose level of phosphorylation is coupled to the cell cycle irrespective of peroxisome positioning in the cell. Our findings demonstrate that both organelle positioning and cell cycle progression control the levels of organelle-specific receptors for molecular motors to ultimately achieve an equidistribution of compartments between mother and daughter cells.

Genome-wide analysis of signaling networks regulating fatty acid-induced gene expression and organelle biogenesis.

Reversible phosphorylation is the most common posttranslational modification used in the regulation of cellular processes. This study of phosphatases and kinases required for peroxisome biogenesis is the first genome-wide analysis of phosphorylation events controlling organelle biogenesis. We evaluate signaling molecule deletion strains of the yeast Saccharomyces cerevisiae for presence of a green fluorescent protein chimera of peroxisomal thiolase, formation of peroxisomes, and peroxisome functionality. We find that distinct signaling networks involving glucose-mediated gene repression, derepression, oleate-mediated induction, and peroxisome formation promote stages of the biogenesis pathway. Additionally, separate classes of signaling proteins are responsible for the regulation of peroxisome number and size. These signaling networks specify the requirements of early and late events of peroxisome biogenesis. Among the numerous signaling proteins involved, Pho85p is exceptional, with functional involvements in both gene expression and peroxisome formation. Our study represents the first global study of signaling networks regulating the biogenesis of an organelle.