ABSTRACT
Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the preautophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.
Subject(s)
Flow Cytometry , Peroxisomes , Saccharomycetales , Peroxisomes/metabolism , Peroxisomes/genetics , Saccharomycetales/genetics , Saccharomycetales/metabolism , High-Throughput Screening Assays , Autophagy , Vacuoles/metabolism , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Macroautophagy/geneticsABSTRACT
Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerases TNKS and TNKS2, which bind the peroxisomal membrane protein PEX14. We propose that RNF146 and TNKS/2 regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased TNKS/2 and RNF146-dependent degradation of non-peroxisomal substrates, including the ß-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of ß-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation but also a novel role in bridging peroxisome function with Wnt/ß-catenin signaling during development.
Subject(s)
Axin Protein , Peroxisomes , Ubiquitin-Protein Ligases , Wnt Signaling Pathway , Peroxisomes/metabolism , Peroxisomes/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Humans , Axin Protein/metabolism , Axin Protein/genetics , Membrane Proteins/metabolism , Membrane Proteins/genetics , beta Catenin/metabolism , beta Catenin/genetics , HEK293 Cells , Protein Transport , CRISPR-Cas SystemsABSTRACT
7-Dehydrocholesterol (7-DHC) is widely present in various organisms and is an important precursor of vitamin D3. Despite significant improvements in the biosynthesis of 7-DHC, it remains insufficient to meet the industrial demands. In this study, we reported high-level production of 7-DHC in an industrial Saccharomyces cerevisiae leveraging subcellular organelles. Initially, the copy numbers of DHCR24 were increased in combination with sterol transcriptional factor engineering and rebalanced the redox power of the strain. Subsequently, the effects of compartmentalizing the post-squalene pathway in peroxisomes were validated by assembling various pathway modules in this organelle. Furthermore, several peroxisomes engineering was conducted to enhance the production of 7-DHC. Utilizing the peroxisome as a vessel for partial post-squalene pathways, the potential of yeast for 7-dehydrocholesterol production was demonstrated by achieving a 26-fold increase over the initial production level. 7-DHC titer reached 640.77 mg/L in shake flasks and 4.28 g/L in a 10 L bench-top fermentor, the highest titer ever reported. The present work lays solid foundation for large-scale and cost-effective production of 7-DHC for practical applications.
Subject(s)
Dehydrocholesterols , Metabolic Engineering , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Dehydrocholesterols/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Peroxisomes/metabolism , Peroxisomes/genetics , DiploidyABSTRACT
Rhodotorula toruloides is a potential workhorse for production of various value-added chemicals including terpenoids, oleo-chemicals, and enzymes from low-cost feedstocks. However, the limited genetic toolbox is hindering its metabolic engineering. In the present study, four type I and one novel type II peroxisomal targeting signal (PTS1/PTS2) were characterized and employed for limonene production for the first time in R. toruloides. The implant of the biosynthesis pathway into the peroxisome led to 111.5 mg/L limonene in a shake flask culture. The limonene titer was further boosted to 1.05 g/L upon dual-metabolic regulation in the cytoplasm and peroxisome, which included employing the acetoacetyl-CoA synthase NphT7, adding an additional copy of native ATP-dependent citrate lyase, etc. The final yield was 0.053 g/g glucose, which was the highest ever reported. The newly characterized PTSs should contribute to the expansion of genetic toolboxes forR. toruloides. The results demonstrated that R. toruloides could be explored for efficient production of terpenoids.
Subject(s)
Cytoplasm , Limonene , Metabolic Engineering , Peroxisomes , Rhodotorula , Limonene/metabolism , Rhodotorula/metabolism , Rhodotorula/genetics , Metabolic Engineering/methods , Peroxisomes/metabolism , Peroxisomes/genetics , Cytoplasm/metabolism , Terpenes/metabolismABSTRACT
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.
Subject(s)
Membrane Proteins , Mitochondria , Peroxisomes , Humans , Mitochondria/metabolism , Mitochondria/genetics , Membrane Proteins/metabolism , Membrane Proteins/genetics , HEK293 Cells , Peroxisomes/metabolism , Peroxisomes/genetics , Peroxins/metabolism , Peroxins/genetics , Protein Transport , Lipoproteins , Repressor ProteinsABSTRACT
Fusarium crown rot, caused by Fusarium pseudograminearum, is a devastating disease affecting the yield and quality of cereal crops. Peroxisomes are single-membrane organelles that play a critical role in various biological processes in eukaryotic cells. To functionally characterise peroxisome biosynthetic receptor proteins FpPEX5 and FpPEX7 in F. pseudograminearum, we constructed deletion mutants, ΔFpPEX5 and ΔFpPEX7, and complementary strains, ΔFpPEX5-C and ΔFpPEX7-C, and analysed the functions of FpPEX5 and FpPEX7 proteins using various phenotypic observations. The deletion of FpPEX5 and FpPEX7 resulted in a significant deficiency in mycelial growth and conidiation and blocked the peroxisomal targeting signal 1 and peroxisomal targeting signal 2 pathways, which are involved in peroxisomal matrix protein transport, increasing the accumulation of lipid droplets and reactive oxygen species. The deletion of FpPEX5 and FpPEX7 may reduce the formation of toxigenic bodies and decrease the pathogenicity of F. pseudograminearum. These results indicate that FpPEX5 and FpPEX7 play vital roles in the growth, asexual reproduction, virulence, and fatty acid utilisation of F. pseudograminearum. This study provides a theoretical basis for controlling stem rot in wheat.
Subject(s)
Fungal Proteins , Fusarium , Peroxisomes , Fusarium/pathogenicity , Fusarium/genetics , Fusarium/metabolism , Fusarium/growth & development , Fungal Proteins/genetics , Fungal Proteins/metabolism , Virulence/genetics , Peroxisomes/metabolism , Peroxisomes/genetics , Trichothecenes/metabolism , Plant Diseases/microbiology , Spores, Fungal/growth & development , Triticum/microbiology , Reactive Oxygen Species/metabolism , Peroxisome-Targeting Signal 1 Receptor/genetics , Peroxisome-Targeting Signal 1 Receptor/metabolism , Gene Deletion , Gene Expression Regulation, Fungal , Peroxisomal Targeting Signal 2 Receptor , Mycelium/growth & development , Mycelium/metabolismABSTRACT
Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on ß-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.
Subject(s)
Metabolic Engineering , Peroxisomes , Peroxisomes/metabolism , Peroxisomes/genetics , Metabolic Engineering/methods , Peroxisomal Targeting Signals/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Pichia/genetics , Pichia/metabolism , Saccharomycetales/genetics , Saccharomycetales/metabolismABSTRACT
The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a genome-wide gRNA library, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.
Subject(s)
Dyneins , Microtubules , Humans , Dyneins/genetics , Microtubules/genetics , Peroxisomes/genetics , CRISPR-Cas Systems , Genetic TechniquesABSTRACT
Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we functionally characterized a putative Trypanosoma PEX1 orthologue by bioinformatic and experimental approaches and show that it is a true PEX1 orthologue. Using yeast two-hybrid analysis, we demonstrate that TbPEX1 can bind to TbPEX6. Endogenously tagged TbPEX1 localizes to glycosomes in the T. brucei parasites. Depletion of PEX1 gene expression by RNA interference causes lethality to the bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion. TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptors TbPEX5 and TbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinated TbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggest that these parasites possess a highly efficient quality control mechanism that exports the import receptors from glycosomes to the cytosol in the absence of a functional TbPEX1-TbPEX6 complex.
Subject(s)
Parasites , Saccharomyces cerevisiae Proteins , Trypanosoma , Animals , Parasites/metabolism , Saccharomyces cerevisiae/metabolism , Peroxisomes/genetics , Peroxisomes/metabolism , Microbodies , ATPases Associated with Diverse Cellular Activities/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Low-grade glioma (LGG), a common primary tumor, mainly originates from astrocytes and oligodendrocytes. Increasing evidence has shown that peroxisomes function in the regulation of tumorigenesis and development of cancer. However, the prognostic value of peroxisome-related genes (PRGs) in LGG has not been reported. Therefore, it is necessary to construct a prognostic risk model for LGG patients based on the expression profiles of peroxisome-related genes. Our study mainly concentrated on developing a peroxisome-related gene signature for overall survival (OS) prediction in LGG patients. First, according to these peroxisome-related genes, all LGG patients from The Cancer Genome Atlas (TCGA) database could be divided into two subtypes. Univariate Cox regression analysis was used to find prognostic peroxisome-related genes in TCGA_LGG dataset, and least absolute shrinkage and selection operator Cox regression analysis was employed to establish a 14-gene signature. The risk score based on the signature was positively associated with unfavorable prognosis. Then, multivariate Cox regression incorporating additional clinical characteristics showed that the 14-gene signature was an independent predictor of LGG. Time-dependent ROC curves revealed good performance of this prognostic signature in LGG patients. The performance about predicting OS of LGG was validated using the GSE107850 dataset derived from the Gene Expression Omnibus (GEO) database. Furethermore, we constructed a nomogram model based on the gene signature and age, which showed a better prognostic power. Gene ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) analyses showed that neuroactive ligand-receptor interaction and phagosome were enriched and that the immune status was decreased in the high-risk group. Finally, cell counting kit-8 (CCK8) were used to detect cell proliferation of U251 and A172 cells. Inhibition of ATAD1 (ATPase family AAA domain-containing 1) and ACBD5 (Acyl-CoA binding-domain-containing-5) expression led to significant inhibition of U251 and A172 cell proliferation. Flow cytometry detection showed that ATAD1 and ACBD5 could induce apoptosis of U251 and A172 cells. Therefore, through bioinformatics methods and cell experiments, our study developed a new peroxisome-related gene signature that migh t help improve personalized OS prediction in LGG patients.
Subject(s)
Glioma , Peroxisomes , Humans , Peroxisomes/genetics , Glioma/genetics , AAA Domain , Adenosine Triphosphatases , Apoptosis , Tumor Microenvironment/geneticsABSTRACT
Peroxisomes are primarily studied in the brain, kidney, and liver due to the conspicuous tissue-specific pathology of peroxisomal biogenesis disorders. In contrast, little is known about the role of peroxisomes in other tissues such as the heart. In this meta-analysis, we explore mitochondrial and peroxisomal gene expression on RNA and protein levels in the brain, heart, kidney, and liver, focusing on lipid metabolism. Further, we evaluate a potential developmental and heart region-dependent specificity of our gene set. We find marginal expression of the enzymes for peroxisomal fatty acid oxidation in cardiac tissue in comparison to the liver or cardiac mitochondrial ß-oxidation. However, the expression of peroxisome biogenesis proteins in the heart is similar to other tissues despite low levels of peroxisomal fatty acid oxidation. Strikingly, peroxisomal targeting signal type 2-containing factors and plasmalogen biosynthesis appear to play a fundamental role in explaining the essential protective and supporting functions of cardiac peroxisomes.
Subject(s)
Peroxisomal Disorders , Peroxisomes , Humans , Peroxisomes/genetics , Peroxisomes/metabolism , Fatty Acids/metabolism , Peroxisomal Disorders/genetics , Peroxisomal Disorders/metabolism , Mitochondria/metabolism , Oxidation-ReductionABSTRACT
The enhanced response of glucagon and its Drosophila homolog, adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous studies have characterized regulatory components transducing linear Akh signaling promoting carbohydrate production, the spatial elucidation of Akh action at the organelle level still remains largely unclear. In this study, we find that Akh phosphorylates extracellular signal-regulated kinase (ERK) and translocates it to peroxisome via calcium/calmodulin-dependent protein kinase II (CaMKII) cascade to increase carbohydrate production in the fat body, leading to hyperglycemia. The mechanisms include that ERK mediates fat body peroxisomal conversion of amino acids into carbohydrates for gluconeogenesis in response to Akh. Importantly, Akh receptor (AkhR) or ERK deficiency, importin-associated ERK retention from peroxisome, or peroxisome inactivation in the fat body sufficiently alleviates high-sugar-diet-induced hyperglycemia. We also observe mammalian glucagon-induced hepatic ERK peroxisomal translocation in diabetic subjects. Therefore, our results conclude that the Akh/glucagon-peroxisomal-ERK axis is a key spatial regulator of glycemic control.
Subject(s)
Drosophila Proteins , Drosophila , Extracellular Signal-Regulated MAP Kinases , Glucagon , Hyperglycemia , Animals , Carbohydrates , Drosophila/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Glucagon/metabolism , Glycemic Control , Peroxisomes/genetics , Peroxisomes/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolismABSTRACT
ß-oxidation of fatty acids is an important metabolic pathway and is a shared function between mitochondria and peroxisomes in mammalian cells. On the other hand, peroxisomes are the sole site for the degradation of fatty acids in yeast. The first reaction of this pathway is catalyzed by the enzyme acyl CoA oxidase housed in the matrix of peroxisomes. Studies in various model organisms have reported the conserved function of the protein in fatty acid oxidation. The importance of this enzyme is highlighted by the lethal conditions caused in humans due to its altered function. In this review, we discuss various aspects ranging from gene expression, structure, folding, and import of the protein in both yeast and human cells. Further, we highlight recent findings on the role of the protein in human health and aging, and discuss the identified mutations in the protein associated with debilitating conditions in patients.
Subject(s)
Peroxisomes , Saccharomyces cerevisiae , Animals , Humans , Acyl-CoA Oxidase/metabolism , Saccharomyces cerevisiae/metabolism , Peroxisomes/genetics , Peroxisomes/metabolism , Oxidation-Reduction , Fatty Acids/metabolism , MammalsABSTRACT
PURPOSE: Zellweger spectrum disorders (ZSDs) are known as autosomal recessive disorders caused by defective peroxisome biogenesis due to bi-allelic pathogenic variants in any of at least 13 different PEX genes. Here, we report 2 unrelated patients who present with an autosomal dominant ZSD. METHODS: We performed biochemical and genetic studies in blood and skin fibroblasts of the patients and demonstrated the pathogenicity of the identified PEX14 variants by functional cell studies. RESULTS: We identified 2 different single heterozygous de novo variants in the PEX14 genes of 2 patients diagnosed with ZSD. Both variants cause messenger RNA mis-splicing, leading to stable expression of similar C-terminally truncated PEX14 proteins. Functional studies indicated that the truncated PEX14 proteins lost their function in peroxisomal matrix protein import and cause increased degradation of peroxisomes, ie, pexophagy, thus exerting a dominant-negative effect on peroxisome functioning. Inhibition of pexophagy by different autophagy inhibitors or genetic knockdown of the peroxisomal autophagy receptor NBR1 resulted in restoration of peroxisomal functions in the patients' fibroblasts. CONCLUSION: Our finding of an autosomal dominant ZSD expands the genetic repertoire of ZSDs. Our study underscores that single heterozygous variants should not be ignored as possible genetic cause of diseases with an established autosomal recessive mode of inheritance.
Subject(s)
Zellweger Syndrome , Humans , Alleles , Peroxisomes/genetics , Peroxisomes/metabolism , Protein Transport/physiology , Proteins/genetics , Zellweger Syndrome/geneticsABSTRACT
Sterol regulatory element-binding protein, Sre1, regulates sterol biosynthesis, lipid metabolism, hypoxia adaptation, and virulence in some fungi, even though its roles are varied in fungal species. However, few studies report its other functions in fungi. Here, we report novel roles of Sre1 homolog, BbSre1, in the insect fungal pathogen, Beauveria bassiana, that regulates oxidative stress response, peroxisome division, and redox homeostasis. The gene disruption stain showed increased sensitivity to oxidative stress, which was in line with oxidative stress-induced-BbSre1 nuclear import and control of antioxidant and detoxification-involved genes. The gene mutation also inhibited peroxisome division, affected redox homeostasis, and impaired lipid/fatty acid metabolism and sterol biosynthesis, which was verified by downregulation of their associated genes. These data broaden our understanding of role of Sre1, which regulates peroxisome division, antioxidant, and detoxification-involved genes for control of redox homeostasis and oxidative stress response that links to lipid/fatty acid metabolism and sterol biosynthesis.
Subject(s)
Antioxidants , Sterol Regulatory Element Binding Proteins , Sterol Regulatory Element Binding Proteins/genetics , Sterol Regulatory Element Binding Proteins/metabolism , Antioxidants/metabolism , Peroxisomes/genetics , Peroxisomes/metabolism , Homeostasis , Oxidative Stress , Oxidation-Reduction , Sterols/metabolism , Fatty Acids/metabolism , LipidsABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters , Saccharomyces cerevisiae Proteins , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Peroxisomes/genetics , Peroxisomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Membrane Proteins/genetics , Membrane Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Peroxins/genetics , Peroxins/metabolismABSTRACT
PCR-based gene targeting enables rapid alteration of the Saccharomyces cerevisiae genome. Here we describe how this method can be applied for directed gene deletions, epitope and fluorescence protein tagging, and conditional gene expression, with a specific focus on peroxisomal proteins.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Peroxisomes/genetics , Peroxisomes/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Polymerase Chain Reaction/methods , Gene TargetingABSTRACT
The development and application of the CRISPR-Cas9 technology for genome editing of mammalian cells have opened up a wealth of possibilities for genetically modifying and manipulating human cells, and use in functional studies or therapeutic approaches.Here we describe the approach that we have been using successfully to generate multiple human cell lines with targeted (partial) gene deletions, i.e., knockout cells, or human cells with modified genomic nucleotide sequences, i.e., knock-in cells, in genes encoding known or putative proteins involved in peroxisome biogenesis or peroxisomal functions.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Animals , Humans , CRISPR-Cas Systems/genetics , Peroxisomes/genetics , Genome , Base Sequence , Mammals/geneticsABSTRACT
Peroxisomes are multifunctional, ubiquitous, and dynamic organelles. They are responsible for diverse metabolic and physiological functions and communicate with other organelles, including the ER, mitochondria, lipid droplets, and lysosomes, through membrane contact sites. However, despite their importance for healthy cell function, remarkably, little is known about how peroxisomes and peroxisomal proteins are regulated under physiological conditions in human cells. Here, we present a method to generate reporter cell lines to measure endogenous expression of peroxisomal proteins of interest. By CRISPR-mediated knock-in of an easily detectable protein-coding tag in-frame into the relevant genomic loci, endogenous levels of the protein of interest in a cell population can be quantified in a high-throughput manner under different conditions. This has important implications for the fundamental understanding of how peroxisomal proteins are regulated and may reveal the therapeutic potential of modulating peroxisomal protein expression to improve cell performance.
Subject(s)
Membrane Proteins , Mitochondria , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Cell Line , Peroxisomes/genetics , Peroxisomes/metabolismABSTRACT
For the biogenesis and maintenance of peroxisomes several proteins, called peroxins, are essential. Malfunctions of these proteins lead to severe diseases summarized as peroxisome biogenesis disorders. The different genetic background of patient-derived cell lines and the residual expression of mutated PEX genes impede analysis of the whole spectrum of cellular functions of affected peroxins. To overcome these difficulties, we have generated a selected PEX knockout resource of HEK T-REx293 cells using the CRISPR/Cas9 technique. Comparative analyses of whole cell lysates revealed PEX-KO specific alterations in the steady-state level of peroxins and variations in the import efficacy of matrix proteins with a Type 2 peroxisomal targeting signal. One of the observed differences concerned PEX1 as in the complete absence of the protein, the number of peroxisomal ghosts is significantly increased. Upon expression of PEX1, import competence and abundance of peroxisomes was adjusted to the level of normal HEK cells. In contrast, expression of an alternatively spliced PEX1 isoform lacking 321 amino acids of the N-terminal region failed to rescue the peroxisomal import defects but reduced the number of peroxisomal vesicles. All in all, the data suggest a novel 'moonlighting' function of human PEX1 in the regulation of pre-peroxisomal vesicles.