RESUMO
Peroxisomes are dynamic organelles with important metabolic functions. Yeast Pex3 is a multifunctional membrane protein aiding in peroxisomal biogenesis, inheritance, and degradation (pexophagy), by interacting with process-specific factors. Using multicolor (live-cell) stimulated emission depletion (STED) nanoscopy, we studied the localization of Pex3 and its binding partners in Hansenula polymorpha. Unlike confocal microscopy, STED allows resolving the membrane of tiny peroxisomes, enabling accurate measurements of the size of all Pex3-labeled peroxisomes. We localized Pex3 and its binding partners at peroxisome-repressing and -inducing conditions and during pexophagy. In-depth quantitative analysis of Pex3 and pexophagy receptor Atg30 showed dynamic changes in their (co)localization. One remarkable response of Atg30 was the shift in position from being sandwiched between clustered peroxisomes at proliferation conditions, to the cytosolically exposed parts of peroxisome clusters upon pexophagy induction. Summarizing, we show that STED allows characterizing dynamics of the localization of peroxisomal proteins in yeast cells.
RESUMO
Peroxisomes are ubiquitous cell organelles involved in various metabolic pathways. In order to properly function, several cofactors, substrates and products of peroxisomal enzymes need to pass the organellar membrane. So far only a few transporter proteins have been identified. We analysed peroxisomal membrane fractions purified from the yeast Hansenula polymorpha by untargeted label-free quantitation mass spectrometry. As expected, several known peroxisome-associated proteins were enriched in the peroxisomal membrane fraction. In addition, several other proteins were enriched, including mitochondrial transport proteins. Localization studies revealed that one of them, the mitochondrial phosphate carrier Mir1, has a dual localization on mitochondria and peroxisomes. To better understand the molecular mechanisms of dual sorting, we localized Mir1 in cells lacking Pex3 or Pex19, two peroxins that play a role in targeting of peroxisomal membrane proteins. In these cells Mir1 only localized to mitochondria, indicating that Pex3 and Pex19 are required to sort Mir1 to peroxisomes. Analysis of the localization of truncated versions of Mir1 in wild-type H. polymorpha cells revealed that most of them localized to mitochondria, but only one, consisting of the transmembrane domains 3-6, was peroxisomal. Peroxisomal localization of this construct was lost in a MIR1 deletion strain, indicating that full-length Mir1 was required for the localization of the truncated protein to peroxisomes. Our data suggest that only full-length Mir1 sorts to peroxisomes, while Mir1 contains multiple regions with mitochondrial sorting information. Data are available via ProteomeXchange with identifier PXD050324.
Assuntos
Proteínas Fúngicas , Mitocôndrias , Peroxissomos , Pichia , Peroxissomos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Pichia/metabolismo , Pichia/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Peroxinas/metabolismo , Peroxinas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Transporte ProteicoRESUMO
Peroxisome biogenesis disorders are caused by pathogenic variants in genes involved in biogenesis and maintenance of peroxisomes. However, mitochondria are also often affected in these diseases. Peroxisomal membrane proteins, including PEX14, have been found to mislocalise to mitochondria in cells lacking peroxisomes. Recent studies indicated that this mislocalisation contributes to mitochondrial abnormalities in PEX3-deficient patient fibroblasts cells. Here, we studied whether mitochondrial morphology is also affected in PEX3-deficient HEK293 cells and whether PEX14 mislocalises to mitochondria in these cells. Using high-resolution imaging techniques, we show that although endogenous PEX14 mislocalises to mitochondria, mitochondrial morphology was normal in PEX3-KO HEK293 cells. However, we discovered that overexpression of tagged PEX14 in wild-type HEK293 cells resulted in its mitochondrial localisation, accompanied by altered mitochondrial morphology. Our data indicate that overexpression of tagged PEX14 alone directly or indirectly cause mitochondrial abnormalities in cells containing peroxisomes.
Assuntos
Proteínas de Membrana , Mitocôndrias , Peroxissomos , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Células HEK293 , Peroxissomos/metabolismo , Peroxissomos/genética , Peroxinas/metabolismo , Peroxinas/genética , Transporte Proteico , Lipoproteínas , Proteínas RepressorasRESUMO
Membrane contact sites are defined as regions of close proximity between two membranes; this association is mediated by protein-protein and/or protein-lipid interactions. Contact sites are often involved in lipid transport, but also can perform other functions. Peroxisomal membrane contact sites have obtained little attention compared to those of other cell organelles. However, recent studies resulted in a big leap in our knowledge of the occurrence, composition and function of peroxisomal contact sites. Studies in yeast strongly contributed to this progress. In this Review, we present an overview of our current knowledge on peroxisomal membrane contact sites in various yeast species, including Hansenula polymorpha, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica. Yeast peroxisomes form contacts with almost all other cellular organelles and with the plasma membrane. The absence of a component of a yeast peroxisomal contact site complex results in a range of peroxisomal phenotypes, including metabolic and biogenesis defects and alterations in organelle number, size or position.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Peroxissomos/metabolismo , Membranas Mitocondriais/metabolismo , Transporte Biológico , Lipídeos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The mechanism behind peroxisomal membrane protein targeting is still poorly understood, with only two yeast proteins believed to be involved and no consensus targeting sequence. Pex19 is thought to bind peroxisomal membrane proteins in the cytosol, and is subsequently recruited by Pex3 at the peroxisomal surface, followed by protein insertion via a mechanism that is as-yet-unknown. However, some peroxisomal membrane proteins still correctly sort in the absence of Pex3 or Pex19, suggesting that multiple sorting pathways exist. Here, we studied sorting of yeast peroxisomal ABC transporter Pxa1. Co-localisation analysis of Pxa1-GFP in a collection of 86 peroxisome-related deletion strains revealed that Pxa1 sorting requires Pex3 and Pex19, while none of the other 84 proteins tested were essential. To identify regions with peroxisomal targeting information in Pxa1, we developed a novel in vivo re-targeting assay, using a reporter consisting of the mitochondrial ABC transporter Mdl1 lacking its N-terminal mitochondrial targeting signal. Using this assay, we showed that the N-terminal 95 residues of Pxa1 are sufficient for retargeting this reporter to peroxisomes. Interestingly, truncated Pxa1 lacking residues 1-95 still localised to peroxisomes. This was confirmed via localisation of various Pxa1 truncation and deletion constructs. However, localisation of Pxa1 lacking residues 1-95 depended on the presence of its interaction partner Pxa2, indicating that this truncated protein does not contain a true targeting signal.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Saccharomyces cerevisiae , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Peroxissomos/genética , Peroxissomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Peroxinas/genética , Peroxinas/metabolismoRESUMO
Correlative light and electron microscopy (CLEM) combines the advantages of protein localization by fluorescence microscopy with the high resolution of electron microscopy. Here, we describe a protocol that we developed for yeast peroxisome research. First, cells are fixed, using conditions that preserve the properties of fluorescent proteins and avoid the introduction of autofluorescence. Next, cryosections are prepared and imaged by fluorescence microscopy. The same sections are used for electron microscopy. Both images are aligned and merged, allowing to localize fluorescent proteins in electron microscopy images. This method was successfully used for peroxisomal membrane contact site research and allows to precisely localize contact site resident proteins at regions where membranes are closely associated at distances far below the resolution of conventional fluorescence microscopy.
Assuntos
Peroxissomos , Proteínas , Microscopia Eletrônica , Saccharomyces cerevisiae , Microscopia de Fluorescência/métodosRESUMO
In the yeast Hansenula polymorpha, the ER protein Pex32 is required for associating peroxisomes to the ER. Here, we report on a structure-function analysis of Pex32. Localization studies of various Pex32 truncations showed that the N-terminal transmembrane domain of Pex32 is responsible for sorting. Moreover, this part of the protein is sufficient for the function of Pex32 in peroxisome biogenesis. The C-terminal DysF domain is required for concentrating Pex32 at ER-peroxisome contact sites and has the ability to bind to peroxisomes. In order to better understand the role of Pex32 in peroxisome biogenesis, we analyzed various peroxisomal proteins in pex32 cells. This revealed that Pex11 levels are strongly reduced in pex32 cells. This may explain the strong reduction in peroxisome numbers in pex32 cells, which also occurs in cells lacking Pex11.
RESUMO
In the yeast Hansenula polymorpha the peroxisomal membrane protein Pex11 and three endoplasmic reticulum localized proteins of the Pex23 family (Pex23, Pex24 and Pex32) are involved in the formation of peroxisome-ER contact sites. Previous studies suggested that these contacts are involved in non-vesicular lipid transfer and important for expansion of the peroxisomal membrane. The absence of Pex32 results in a severe peroxisomal phenotype, while cells lacking Pex11, Pex23 or Pex24 show milder defects and still are capable to form peroxisomes and grow on methanol. We performed transposon mutagenesis on H. polymorpha pex11 cells and selected mutants that lost the capacity to grow on methanol and are severely blocked in peroxisome formation. This strategy resulted in the identification of Vps13, a highly conserved contact site protein involved in bulk lipid transfer. Our data show that peroxisome formation and function is normal in cells of a vps13 single deletion strain. However, Vps13 is essential for peroxisome biogenesis in pex11. Notably, Vps13 is also required for peroxisome formation in pex23 and pex24 cells. These data suggest that Vps13 is crucial for peroxisome formation in cells with reduced peroxisome-endoplasmic reticulum contact sites and plays a redundant function in lipid transfer from the ER to peroxisomes.
RESUMO
PEX genes encode proteins involved in peroxisome biogenesis and proliferation. Using a comparative genomics approach, we clarify the evolutionary relationships between the 37 known PEX proteins in a representative set of eukaryotes, including all common model organisms, pathogenic unicellular eukaryotes and human. A large number of previously unknown PEX orthologs were identified. We analyzed all PEX proteins, their conservation and domain architecture and defined the core set of PEX proteins that is required to make a peroxisome. The molecular processes in peroxisome biogenesis in different organisms were put into context, showing that peroxisomes are not static organelles in eukaryotic evolution. Organisms that lack peroxisomes still contain a few PEX proteins, which probably play a role in alternative processes. Finally, the relationships between PEX proteins of two large families, the Pex11 and Pex23 families, were analyzed, thereby contributing to the understanding of their complicated and sometimes incorrect nomenclature. We provide an exhaustive overview of this important eukaryotic organelle.
RESUMO
Retention of peroxisomes in yeast mother cells requires Inp1, which is recruited to the organelle by the peroxisomal membrane protein Pex3. Here we show that Hansenula polymorpha Inp1 associates peroxisomes to the plasma membrane. Peroxisome-plasma membrane contact sites disappear upon deletion of INP1 but increase upon INP1 overexpression. Analysis of truncated Inp1 variants showed that the C terminus is important for association to the peroxisome, while a stretch of conserved positive charges and a central pleckstrin homology-like domain are important for plasma membrane binding. In cells of a PEX3 deletion, strain Inp1-GFP localizes to the plasma membrane, concentrated in patches near the bud neck and in the cortex of nascent buds. Upon disruption of the actin cytoskeleton by treatment of the cells with latrunculin A, Inp1-GFP became cytosolic, indicating that Inp1 localization is dependent on the presence of an intact actin cytoskeleton.
Assuntos
Proteínas de Membrana/genética , Peroxinas/genética , Peroxissomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Citoesqueleto de Actina/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Membrana Celular/genética , Retículo Endoplasmático/genética , Regulação Fúngica da Expressão Gênica/genética , Membranas Mitocondriais/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Tiazolidinas/farmacologiaRESUMO
The yeast Hansenula polymorpha contains four members of the Pex23 family of peroxins, which characteristically contain a DysF domain. Here we show that all four H. polymorpha Pex23 family proteins localize to the endoplasmic reticulum (ER). Pex24 and Pex32, but not Pex23 and Pex29, predominantly accumulate at peroxisome-ER contacts. Upon deletion of PEX24 or PEX32 - and to a much lesser extent, of PEX23 or PEX29 - peroxisome-ER contacts are lost, concomitant with defects in peroxisomal matrix protein import, membrane growth, and organelle proliferation, positioning and segregation. These defects are suppressed by the introduction of an artificial peroxisome-ER tether, indicating that Pex24 and Pex32 contribute to tethering of peroxisomes to the ER. Accumulation of Pex32 at these contact sites is lost in cells lacking the peroxisomal membrane protein Pex11, in conjunction with disruption of the contacts. This indicates that Pex11 contributes to Pex32-dependent peroxisome-ER contact formation. The absence of Pex32 has no major effect on pre-peroxisomal vesicles that occur in pex3 atg1 deletion cells.
Assuntos
Peroxissomos , Proteínas de Saccharomyces cerevisiae , Retículo Endoplasmático/genética , Proteínas de Membrana/genética , Biogênese de Organelas , Peroxinas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , SaccharomycetalesRESUMO
The bacterial plasma membrane is an important cellular compartment. In recent years it has become obvious that protein complexes and lipids are not uniformly distributed within membranes. Current hypotheses suggest that flotillin proteins are required for the formation of complexes of membrane proteins including cell-wall synthetic proteins. We show here that bacterial flotillins are important factors for membrane fluidity homeostasis. Loss of flotillins leads to a decrease in membrane fluidity that in turn leads to alterations in MreB dynamics and, as a consequence, in peptidoglycan synthesis. These alterations are reverted when membrane fluidity is restored by a chemical fluidizer. In vitro, the addition of a flotillin increases membrane fluidity of liposomes. Our data support a model in which flotillins are required for direct control of membrane fluidity rather than for the formation of protein complexes via direct protein-protein interactions.
Every living cell is enclosed by a flexible membrane made of molecules known as phospholipids, which protects the cell from harmful chemicals and other threats. In bacteria and some other organisms, a rigid structure known as the cell wall sits just outside of the membrane and determines the cell's shape. There are several proteins in the membrane of bacteria that allow the cell to grow by assembling new pieces of the cell wall. To ensure these proteins expand the cell wall at the right locations, another protein known as MreB moves and organizes them to the appropriate place in the membrane and controls their activity. Previous studies have found that another class of proteins called flotillins are involved in arranging proteins and phospholipid molecules within membranes. Bacteria lacking these proteins do not grow properly and are unable to maintain their normal shape. However, the precise role of the flotillins remained unclear. Here, Zielinska, Savietto et al. used microscopy approaches to study flotillins in a bacterium known as Bacillus subtilis. The experiments found that, in the presence of flotillins, MreB moved around the membrane more quickly (suggesting it was more active) than when no flotillins were present. Similar results were observed when bacterial cells lacking flotillins were treated with a chemical that made membranes more 'fluid' that is, made it easier for the molecules within the membrane to travel around. Further experiments found that flotillins allowed the phospholipid molecules within an artificial membrane to move around more freely, which increases the fluidity of the membrane. These findings suggest that flotillins make the membranes of bacterial cells more fluid to help cells expand their walls and perform several other processes. Understanding how bacteria control the components of their membranes will further our understanding of how many currently available antibiotics work and may potentially lead to the design of new antibiotics in the future.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/fisiologia , Fluidez de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Peptidoglicano/biossínteseRESUMO
Here, we describe a novel peroxin, Pex37, in the yeast Hansenula polymorpha. H. polymorpha Pex37 is a peroxisomal membrane protein, which belongs to a protein family that includes, among others, the Neurospora crassa Woronin body protein Wsc, the human peroxisomal membrane protein PXMP2, the Saccharomyces cerevisiae mitochondrial inner membrane protein Sym1, and its mammalian homologue MPV17. We show that deletion of H. polymorpha PEX37 does not appear to have a significant effect on peroxisome biogenesis or proliferation in cells grown at peroxisome-inducing growth conditions (methanol). However, the absence of Pex37 results in a reduction in peroxisome numbers and a defect in peroxisome segregation in cells grown at peroxisome-repressing conditions (glucose). Conversely, overproduction of Pex37 in glucose-grown cells results in an increase in peroxisome numbers in conjunction with a decrease in their size. The increase in numbers in PEX37-overexpressing cells depends on the dynamin-related protein Dnm1. Together our data suggest that Pex37 is involved in peroxisome fission in glucose-grown cells. Introduction of human PXMP2 in H. polymorpha pex37 cells partially restored the peroxisomal phenotype, indicating that PXMP2 represents a functional homologue of Pex37. H.polymorpha pex37 cells did not show aberrant growth on any of the tested carbon and nitrogen sources that are metabolized by peroxisomal enzymes, suggesting that Pex37 may not fulfill an essential function in transport of these substrates or compounds required for their metabolism across the peroxisomal membrane.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Organelas/metabolismo , Peroxissomos/metabolismo , Saccharomycetales/química , Proteínas Fúngicas/química , Proteínas de Membrana/química , Organelas/química , Peroxissomos/química , Saccharomycetales/citologia , Saccharomycetales/metabolismoRESUMO
There is an ongoing debate on how peroxisomes form: by growth and fission of pre-existing peroxisomes or de novo from another membrane. It has been proposed that, in wild type yeast cells, peroxisome fission and careful segregation of the organelles over mother cells and buds is essential for organelle maintenance. Using live cell imaging we observed that cells of the yeast Hansenula polymorpha, lacking the peroxisome fission protein Pex11, still show peroxisome fission and inheritance. Also, in cells of mutants without the peroxisome inheritance protein Inp2 peroxisome segregation can still occur. In contrast, peroxisome fission and inheritance were not observed in cells of a pex11 inp2 double deletion strain. In buds of cells of this double mutant, new organelles likely appear de novo. Growth of pex11 inp2 cells on methanol, a growth substrate that requires functional peroxisomes, is retarded relative to the wild type control. Based on these observations we conclude that in H. polymorpha de novo peroxisome formation is a rescue mechanism, which is less efficient than organelle fission and inheritance to maintain functional peroxisomes.
Assuntos
Biogênese de Organelas , Peroxinas/fisiologia , Peroxissomos/fisiologia , Pichia/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Microrganismos Geneticamente Modificados , Mutação , Peroxinas/genética , Pichia/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/fisiologiaRESUMO
The peroxisomal membrane protein (PMP) Pex3 and its cytosolic interaction partner Pex19 have been implicated in peroxisomal membrane biogenesis. Although these peroxins have been extensively studied, no consensus has been reached yet on how they operate. Here, we discuss two major models of their function, namely, in direct insertion of proteins into the peroxisomal membrane or in formation of PMP-containing vesicles from the endoplasmic reticulum (ER). Pex3 can also recruit other proteins to the peroxisomal membrane (e.g., Inp1, Atg30, Atg36), thereby fulfilling roles in other processes such as autophagy and organelle retention. Recent studies indicate that Pex3 and Pex19 can also facilitate sorting of certain membrane proteins to other cellular organelles, including the ER, lipid droplets, and mitochondria.
Assuntos
Lipoproteínas/fisiologia , Proteínas de Membrana/fisiologia , Peroxinas/fisiologia , Autofagia , Retículo Endoplasmático/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Peroxinas/metabolismo , Peroxissomos/metabolismo , Ligação ProteicaRESUMO
Using electron and fluorescence microscopy techniques, we identified various physical contacts between peroxisomes and other cell organelles in the yeast Hansenula polymorpha. In exponential glucose-grown cells, which typically contain a single small peroxisome, contacts were only observed with the endoplasmic reticulum and the plasma membrane. Here we focus on a novel peroxisome-vacuole contact site that is formed when glucose-grown cells are shifted to methanol containing media, conditions that induce strong peroxisome development. At these conditions, the small peroxisomes rapidly increase in size, a phenomenon that is paralleled by the formation of distinct intimate contacts with the vacuole. Localization studies showed that the peroxin Pex3 accumulated in patches at the peroxisome-vacuole contact sites. In wild-type cells growing exponentially on medium containing glucose, peroxisome-vacuole contact sites were never observed. However, upon overproduction of Pex3 peroxisomes also associated to vacuoles at these growth conditions. Our observations strongly suggest a role for Pex3 in the formation of a novel peroxisome-vacuole contact site. This contact likely plays a role in membrane growth as it is formed solely at conditions of strong peroxisome expansion.
Assuntos
Proteínas de Membrana/metabolismo , Peroxinas/metabolismo , Peroxissomos/metabolismo , Pichia/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Membranas Mitocondriais/metabolismo , Peroxissomos/fisiologia , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismoRESUMO
Peroxisomes are single membrane enclosed cell organelles, which are present in almost all eukaryotic cells. In addition to the common peroxisomal pathways such as ß-oxidation of fatty acids and decomposition of H2O2, these organelles fulfil a range of metabolic and non-metabolic functions. Peroxisomes are very important since various human disorders exist that are caused by a defect in peroxisome function. Here we describe our current knowledge on the molecular mechanisms of peroxisome biogenesis in yeast, including peroxisomal protein sorting, organelle dynamics and peroxisomal membrane contact sites.
Assuntos
Peroxissomos/metabolismo , Leveduras/genética , Leveduras/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Metabolismo dos Lipídeos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutação , Biogênese de Organelas , Peroxinas/genética , Peroxinas/metabolismo , Sinais de Orientação para Peroxissomos , Peroxissomos/genética , Peroxissomos/ultraestrutura , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Leveduras/ultraestruturaRESUMO
The multi-subunit mitochondrial contact site and cristae organizing system (MICOS) is a conserved protein complex of the inner mitochondrial membrane that is essential for maintenance of cristae architecture. The core subunit Mic10 forms large oligomers that build a scaffold and induce membrane curvature. The regulation of Mic10 oligomerization is poorly understood. We report that Mic26 exerts a destabilizing effect on Mic10 oligomers and thus functions in an antagonistic manner to the stabilizing subunit Mic27. The mitochondrial signature phospholipid cardiolipin shows a stabilizing function on Mic10 oligomers. Our findings indicate that the Mic10 core machinery of MICOS is regulated by several mechanisms, including interaction with cardiolipin and antagonistic actions of Mic26 and Mic27.
Assuntos
Cardiolipinas/farmacologia , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/química , Proteínas Mitocondriais/química , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Here, we used fluorescence microscopy and a peroxisome-targeted tandem fluorescent protein timer to determine the relative age of peroxisomes in yeast. Our data indicate that yeast cells contain a heterogeneous population of relatively old and young peroxisomes. During budding, the peroxisome retention factor inheritance of peroxisomes protein 1 (Inp1) selectively associates to the older organelles, which are retained in the mother cells. Inp2, a protein required for transport of peroxisomes to the bud, preferentially associates to younger organelles. Using a microfluidics device, we demonstrate that the selective segregation of younger peroxisomes to the buds is carefully maintained during multiple budding events. The replicative lifespan of mother cells increased upon deletion of INP2, which resulted in the retention of all organelles in mother cells. These data suggest that, in wild-type yeast, transport of aged and deteriorated peroxisomes to the bud is prevented, whereas the young and vital organelles are preferably transported to the newly forming buds.