RESUMO
Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles, however, are unclear, mainly because structures of higher-order assemblies are still lacking. Here, using an integrative approach, we provide the structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment, and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20-nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.
RESUMO
The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and non-phosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes in oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Overexpression of Cit2p rescued the growth phenotype of yeast cells expressing Pex14p-S266D in oleic acid. Our data indicate that phosphorylation of Pex14p at S266 provides a mechanism for controlling the peroxisomal import of Cit2p, which helps S. cerevisiae cells to adjust their carbohydrate metabolism according to the nutritional conditions.
RESUMO
The mechanical setup of a novel scanning reflection X-ray microscope is presented. It is based on zone plate optics optimized for reflection mode in the EUV spectral range. The microscope can operate at synchrotron radiation beamlines as well as at laboratory-based plasma light sources. In contrast to established X-ray transmission microscopes that use thin foil samples, the new microscope design presented here allows the investigation of any type of bulk materials. Importantly, this permits the investigation of magnetic materials by employing experimental techniques based on X-ray magnetic circular dichroism, X-ray linear magnetic dichroism or the transversal magneto-optical Kerr effect (T-MOKE). The reliable functionality of the new microscope design has been demonstrated by T-MOKE microscopy spectra of Fe/Cr-wedge/Fe trilayer samples. The spectra were recorded at various photon energies across the Fe 3p edge revealing the orientation of magnetic domains in the sample.
RESUMO
Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Peroxinas/genética , Peroxissomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana Transportadoras/biossíntese , Peroxinas/biossíntese , Peroxissomos/metabolismo , Proteoma/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Vesículas Transportadoras/genética , Vesículas Transportadoras/metabolismo , Enzimas de Conjugação de Ubiquitina/biossíntese , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Reversible protein phosphorylation is a frequently occurring posttranslational modification mediated by protein kinases and phosphatases that plays an essential role in the regulation of a large number of cellular processes. Evidence is accumulating that protein phosphorylation is also an important mechanism governing processes associated with peroxisome biology. For an improved and detailed understanding of these processes and their regulation it is therefore crucial to study phosphorylation of peroxisome-associated proteins and to determine the phosphorylated amino acid(s). To place peroxisome-related processes into a larger, cellular context, it is further required to identify the kinases and phosphatases catalyzing phosphorylation and dephosphorylation events in peroxisomal proteins. We here provide a strategy for the targeted analysis of peroxisomal phosphoproteins of Saccharomyces cerevisiae combining affinity purification of epitope-tagged peroxisomal proteins with Phos-tag SDS-PAGE and high-resolution mass spectrometry (MS) for the identification and precise localization of in vivo phosphosites. Furthermore, we describe a protocol for an MS-based in vitro kinase assay using recombinant peroxisomal proteins and a selected kinase facilitating the site-resolved analysis of kinase-substrate relationships.
Assuntos
Peroxissomos/metabolismo , Proteínas/metabolismo , Proteômica , Cromatografia Líquida , Espectrometria de Massas , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas/isolamento & purificação , Proteólise , Proteômica/métodos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Expression of the archaellum, the archaeal-type IV pilus-like rotating motility structure is upregulated under nutrient limitation. This is controlled by a network of regulators, called the archaellum regulatory network (arn). Several of the components of this network in Sulfolobus acidocaldarius can be phosphorylated, and the deletion of the phosphatase PP2A results in strongly increased motility during starvation, indicating a role for phosphorylation in the regulation of motility. Analysis of the motility of different protein kinase deletion strains revealed that deletion of saci_0965, saci_1181, and saci_1193 resulted in reduced motility, whereas the deletion of saci_1694 resulted in hypermotility. Here ArnC (Saci_1193) and ArnD (Saci_1694) are characterized. Purified ArnC and ArnD phosphorylate serine and threonine residues in the C-terminus of the repressor ArnB. arnC is upregulated in starvation medium, whereas arnD is constitutively expressed. However, while differences in the expression and levels of flaB were observed in the ΔarnD strain during growth under rich conditions, under nutrient limiting conditions the ΔarnC and ΔarnD strains showed no large differences in the expression levels of the archaellum or of the studied regulators. This suggests that next to the regulation via the archaellum regulatory network additional regulatory mechanisms of expression and/or activity of the archaellum exist.
Assuntos
Proteínas Arqueais/metabolismo , Flagelos/metabolismo , Regulação da Expressão Gênica em Archaea , Proteínas Quinases/metabolismo , Sulfolobus acidocaldarius/metabolismo , Proteínas Arqueais/genética , Flagelos/genética , Deleção de Genes , Fosforilação , Domínios Proteicos , Proteínas Quinases/genética , Transdução de Sinais/fisiologia , Inanição , Sulfolobus acidocaldarius/genéticaRESUMO
Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by peroxisomal targeting sequences (PTS). In the following, the assembled receptor-cargo complex is targeted to the peroxisomal membrane where it docks to the docking-complex as part of the peroxisomal translocation machinery. The docking-complex is composed of Pex13p, Pex14p and in yeast also Pex17p, whose function is still elusive. In order to characterize the function of Pex17p, we compared the composition and size of peroxisomal receptor-docking complexes from wild-type and pex17Δ cells. Our data demonstrate that the deficiency of Pex17p affects the stoichiometry of the constituents of an isolated 600kDa complex and that pex17Δ cells lack a high molecular weight complex (>900kDa) of unknown function. We identified the dynein light chain protein Dyn2p as an additional core component of the Pex14p/Pex17p-complex. Both, Pex14p and Pex17p interact directly with Dyn2p, but in vivo, Pex17p turned out to be prerequisite for an association of Dyn2p with Pex14p. Finally, like pex17Δ also dyn2Δ cells lack the high molecular weight complex. As dyn2Δ cells also display reduced peroxisomal function, our data indicate that Dyn2p-dependent formation of the high molecular weight Pex14p-complex is required to maintain peroxisomal function on wild-type level.
Assuntos
Dineínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Complexos Multiproteicos/metabolismo , Peroxissomos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dineínas/genética , Proteínas de Membrana Transportadoras/genética , Complexos Multiproteicos/genética , Peroxinas , Peroxissomos/genética , Transporte Proteico/fisiologia , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Peroxisomes are highly dynamic organelles that can rapidly change in size, abundance, and protein content in response to alterations in nutritional and other environmental conditions. These dynamic changes in peroxisome features, referred to as peroxisome dynamics, rely on the coordinated action of several processes of peroxisome biogenesis. Revealing the regulatory mechanisms of peroxisome dynamics is an emerging theme in cell biology. These mechanisms are inevitably linked to and synchronized with the biogenesis and degradation of peroxisomes. To date, the key players and basic principles of virtually all steps in the peroxisomal life cycle are known, but regulatory mechanisms remained largely elusive. A number of recent studies put the spotlight on reversible protein phosphorylation for the control of peroxisome dynamics and highlighted peroxisomes as hubs for cellular signal integration and regulation. Here, we will present and discuss the results of several studies performed using yeast and mammalian cells that convey a sense of the impact protein phosphorylation may have on the modulation of peroxisome dynamics by regulating peroxisomal matrix and membrane protein import, proliferation, inheritance, and degradation. We further put forward the idea to make use of current data on phosphorylation sites of peroxisomal and peroxisome-associated proteins reported in advanced large-scale phosphoproteomic studies.
Assuntos
Autofagia , Glicerol-3-Fosfato Desidrogenase (NAD+)/metabolismo , Biogênese de Organelas , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Regulação da Expressão Gênica , Glicerol-3-Fosfato Desidrogenase (NAD+)/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/química , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de SinaisRESUMO
Despite their simplicity compared to multicellular organisms, single-celled yeasts such as the baker's yeast Saccharomyces cerevisiae are widely recognized as model organisms for the study of eukaryotic cell biology. To gain deeper insights into the molecular mechanisms underlying cellular processes, it is of utmost interest to establish the interactome of distinct proteins and to thoroughly analyze the composition of individual protein complexes and their dynamics. Combining affinity purification of epitope-tagged proteins with high-resolution mass spectrometry and quantitative proteomics strategies, in particular stable isotope labeling by amino acids in cell culture (SILAC), represents an unbiased and powerful approach for a most accurate characterization of protein complexes. In this chapter, we provide detailed protocols for the generation of yeast strains (S. cerevisiae) amenable to SILAC-labeling, for epitope tagging of a protein of interest for affinity purification, and for the SILAC-based characterization of membrane protein complexes including the identification of stable core components and transient interaction partners.
Assuntos
Aminoácidos/química , Marcação por Isótopo/métodos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteômica/métodos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Métodos Analíticos de Preparação de Amostras , Células Cultivadas , Cromatografia Líquida , Técnicas de Cultura , Genômica , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem , Tripsina/metabolismoRESUMO
Peroxisome biogenesis initiates at the endoplasmic reticulum (ER) and maturation allows for the formation of metabolically active organelles. Yet, peroxisomes can also multiply by growth and division. Several proteins, called peroxins, are known to participate in these processes but little is known about their organization to orchestrate peroxisome proliferation. Here, we demonstrate that regulation of peroxisome proliferation relies on the integrity of the tubular ER network. Using a dual track SILAC-based quantitative interaction proteomics approach, we established a comprehensive network of stable as well as transient interactions of the peroxin Pex30p, an integral membrane protein. Through association with merely ER resident proteins, in particular with proteins containing a reticulon homology domain, and with other peroxins, Pex30p designates peroxisome contact sites at ER subdomains. We show that Pex30p traffics through the ER and segregates in punctae to which peroxisomes specifically append, and we ascertain its transient interaction with all subunits of the COPI coatomer complex suggesting the involvement of a vesicle-mediated transport. We establish that the membrane protein Pex30p facilitates the connection of peroxisomes to the ER. Taken together, our data indicate that Pex30p-containing protein complexes act as focal points from which peroxisomes can form and that the tubular ER architecture organized by the reticulon homology proteins Rtn1p, Rtn2p and Yop1p controls this process.