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1.
Methods Mol Biol ; 2564: 203-211, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107343

RESUMO

Fluorescent proteins (FPs) remarkably advanced the study of cellular biology of plants. The most common application is their use as reporter proteins to determine the subcellular localization of a protein of interest (POI) by endogenous expression of a suitable FP-POI fusion construct in plant cells. In this chapter we describe three approaches, namely, particle bombardment, protoplast transformation, and Agrobacterium infiltration, to transiently express such fusion constructs in plant cells of different species. These approaches are versatile and can be utilized for diverse fluorescent protein-based applications.


Assuntos
Agrobacterium , Plantas , Agrobacterium/genética , Agrobacterium/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Vegetais/metabolismo , Plantas/genética , Plantas/metabolismo , Transporte Proteico
2.
Methods Mol Biol ; 2594: 97-106, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36264491

RESUMO

The transcription factor FoxO1 (forkhead box O1) regulates genes that are involved in development, metabolism, cellular innovation, longevity, and stress responses. Assessment of FoxO1 activity is therefore critical to understand the regulatory network of this transcription factor. FoxO1 transactivation activity relies on its ability to bind to the promoters of target genes, which is controlled by posttranslational modifications (e.g., dephosphorylation or phosphorylation) that may promote nuclear translocation or exclusion of FoxO1. In this chapter we describe the protocols for FoxO1 activity assessment using Western blotting analysis of the posttranslational modification of FoxO1 in whole cell lysates and ELISA of DNA binding activity of FoxO1 in nuclear extracts.


Assuntos
DNA , Fatores de Transcrição Forkhead , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fosforilação/fisiologia , Transporte Proteico , DNA/metabolismo
3.
Gene ; 851: 146971, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36261082

RESUMO

The main function of Sec61 complex is participating in the transport of polypeptide chains across the endoplasmic reticulum. The Sec61α subunit is the largest subunit of the Sec61 complex and shows high degree of conservation. In this study, we identified the NbSec61α and NbSec61γ genes in the microsporidian Nosema bombycis for the first time. Multiple sequence alignment showed that the sequence similarity between NbSec61α and homologous proteins of other microsporidia was greater than 48 %. NbSec61α contains a "plug" domain (amino acids 40-74) unique to the Sec61/SecY complex. Phylogenetic analysis based on NbSec61α and NbSec61γ indicated that the N. bombycis was closely related to Nosema granulosis, Nosema ceranae and Nosema apis. Indirect immunfluorescence assay showed that NbSec61α and NbSec61γ were mainly distributed in the perinuclear region of N. bombycis in different developmental phases. qRT-PCR results revealed that the expression level of NbSec61α gene increased in the early stage and reached the highest at 48 h, then decreased in the late stages. After knockdown of NbSec61α, the expression of NbSec61α, NbSec61γ and NbssrRNA genes were all significantly down-regulated. These results suggest that the NbSec61α and NbSec61γ may play an important role in the intracellular development of N. bombycis.


Assuntos
Bombyx , Nosema , Animais , Filogenia , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Nosema/genética , Nosema/metabolismo , Alinhamento de Sequência , Transporte Proteico , Bombyx/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo
4.
Methods Mol Biol ; 2603: 43-58, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36370269

RESUMO

The protein cargo of extracellular vesicles (EVs) determines their impact on recipient cell types and the downstream effects on biological function. Environmental cues can modify EV loading with proteins derived from the plasma membrane via endocytosis, obtained from the preexisting cytosolic pool via active sorting, or packaging with newly synthesized proteins drawn from trans-golgi networks. Given the major impact these pathways exert on EV content and functional potential, it is important to study how defined stimuli influence protein sorting into these vesicles for dispersal. To this end, pSILAC-based approaches can be used to pulse/trace the origins of EV protein content and thereby provide valuable insight into vesicle biology and likely effects on intercellular communication in diverse settings.


Assuntos
Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Transporte Proteico , Comunicação Celular , Proteínas/metabolismo , Endocitose
5.
Biochim Biophys Acta Proteins Proteom ; 1871(1): 140865, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36272538

RESUMO

A large number of nascent polypeptides have to get across a membrane in targeting to the proper subcellular locations. The SecYEG protein complex, a homolog of the Sec61 complex in eukaryotic cells, has been viewed as the common translocon at the inner membrane for targeting proteins to three extracytoplasmic locations in Gram-negative bacteria, despite the lack of direct verification in living cells. Here, via unnatural amino acid-mediated protein-protein interaction analyses in living cells, in combination with genetic studies, we unveiled a hitherto unreported SecAN protein that seems to be directly involved in translocationg nascent outer membrane proteins across the plasma membrane; it consists of the N-terminal 375 residues of the SecA protein and exists as a membrane-integrated homooligomer. Our new findings place multiple previous observations related to bacterial protein targeting in proper biochemical and evolutionary contexts.


Assuntos
Proteínas de Escherichia coli , Proteínas de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas SecA , Canais de Translocação SEC/genética , Canais de Translocação SEC/química , Canais de Translocação SEC/metabolismo , Transporte Proteico
6.
J Cell Biol ; 222(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36239631

RESUMO

At the trans-Golgi, complex traffic connections exist to the endolysosomal system additional to the main Golgi-to-plasma membrane secretory route. Here, we investigated three hits in a Drosophila screen displaying secretory cargo accumulation in autophagic vesicles: ESCRT-III component Vps20, SNARE-binding Rop, and lysosomal pump subunit VhaPPA1-1. We found that Vps20, Rop, and lysosomal markers localize near the trans-Golgi. Furthermore, we document that the vicinity of the trans-Golgi is the main cellular location for lysosomes and that early, late, and recycling endosomes associate as well with a trans-Golgi-associated degradative compartment where basal microautophagy of secretory cargo and other materials occurs. Disruption of this compartment causes cargo accumulation in our hits, including Munc18 homolog Rop, required with Syx1 and Syx4 for Rab11-mediated endosomal recycling. Finally, besides basal microautophagy, we show that the trans-Golgi-associated degradative compartment contributes to the growth of autophagic vesicles in developmental and starvation-induced macroautophagy. Our results argue that the fly trans-Golgi is the gravitational center of the whole endomembrane system.


Assuntos
Autofagia , Endossomos , Complexo de Golgi , Lisossomos , Animais , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteínas rab de Ligação ao GTP
7.
J Cell Biol ; 222(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36282215

RESUMO

Arl8b, an Arf-like GTP-binding protein, regulates cargo trafficking and positioning of lysosomes. However, it is unknown whether Arl8b regulates lysosomal cargo sorting. Here, we report that Arl8b binds to the Rab4 and Rab14 interaction partner, RUN and FYVE domain-containing protein (RUFY) 1, a known regulator of cargo sorting from recycling endosomes. Arl8b determines RUFY1 endosomal localization through regulating its interaction with Rab14. RUFY1 depletion led to a delay in CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. We identified the dynein-dynactin complex as an RUFY1 interaction partner, and similar to a subset of activating dynein adaptors, the coiled-coil region of RUFY1 was required for interaction with dynein and the ability to mediate dynein-dependent organelle clustering. Our findings suggest that Arl8b and RUFY1 play a novel role on recycling endosomes, from where this machinery regulates endosomes to TGN retrieval of CI-M6PR and, consequently, lysosomal cargo sorting.


Assuntos
Fatores de Ribosilação do ADP , Proteínas Adaptadoras de Transdução de Sinal , Dineínas , Endossomos , Lisossomos , Proteínas rab de Ligação ao GTP , Humanos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Endossomos/metabolismo , Células HeLa , Lisossomos/metabolismo , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo
8.
Life Sci Alliance ; 6(1)2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36253107

RESUMO

Mitochondria play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here, we show that the yeast cytosolic ubiquitin conjugase Ubc8 plays a crucial role in the remodeling process when cells transition from respiratory to fermentative conditions. Ubc8 is a conserved and well-studied component of the catabolite control system that is known to regulate the stability of gluconeogenic enzymes. Unexpectedly, we found that Ubc8 also promotes the assembly of the translocase of the outer membrane of mitochondria (TOM) and increases the levels of its cytosol-exposed receptor subunit Tom22. Ubc8 deficiency results in compromised protein import into mitochondria and reduced steady-state levels of mitochondrial proteins. Our observations show that Ubc8, which is controlled by the prevailing metabolic conditions, promotes the switch from glucose synthesis to glucose usage in the cytosol and induces the biogenesis of the mitochondrial TOM machinery to improve mitochondrial protein import during phases of metabolic transition.


Assuntos
Transporte Proteico , Proteínas de Saccharomyces cerevisiae , Enzimas de Conjugação de Ubiquitina , gama-Glutamil Hidrolase/metabolismo , Glucose/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
9.
Dis Model Mech ; 15(11)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36420970

RESUMO

Many inherited visual diseases arise from mutations that affect the structure and function of photoreceptor cells. In some cases, the pathology is accompanied by a massive release of extracellular vesicles from affected photoreceptors. In this study, we addressed whether vesicular release is an exclusive response to ongoing pathology or a normal homeostatic phenomenon amplified in disease. We analyzed the ultrastructure of normal photoreceptors from both rod- and cone-dominant mammalian species and found that these cells release microvesicles budding from their inner segment compartment. Inner segment-derived microvesicles vary in their content, with some of them containing the visual pigment rhodopsin and others appearing to be interconnected with mitochondria. These data suggest the existence of a fundamental process whereby healthy mammalian photoreceptors release mistrafficked or damaged inner segment material as microvesicles into the interphotoreceptor space. This release may be greatly enhanced under pathological conditions associated with defects in protein targeting and trafficking. This article has an associated First Person interview with the first author of the paper.


Assuntos
Células Fotorreceptoras , Rodopsina , Animais , Humanos , Células Fotorreceptoras/metabolismo , Rodopsina/metabolismo , Transporte Proteico , Mamíferos/metabolismo
10.
PLoS One ; 17(11): e0277811, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36413554

RESUMO

The occurrence of cancer metastasis may be related to stem cells in normal tissues. We searched for patient IDs with both normal tissue stem cell values and TCGA (The Cancer Genome Atlas) clinical data for pairing and obtained 639 sets of data (stemness index of normal tissue, stemness index of tumor tissue, cancer stage, distant metastasis, tumor size) and invasion, and lymph node involvement). However, clinical data on cancer metastasis are of only four stages (e.g., Stage I, II, III, and IV), which cannot show subtle changes continuously. We need to find an effective data mining method to transform this four-valued clinical description into a numerical curve. We data-mine this data through numericalization, sorting, and noise reduction filtering. The results showed that: as the normal tissue stemness value (NS) increased, the tumor tissue stemness value (TS) increased proportionally (1.26 times NS). When NS >0.5, the rate of change in TS decelerated (0.43 times NS), and tumor metastasis began to occur. Clinical indicators, such as cancer stage, distant metastasis, tumor size and invasion, and lymph node involvement, showed that tumor metastasis became more and more severe with the increase of NS. This study suggests that tumor metastasis is triggered when the NS in the patient's body is more significant than 0.5.


Assuntos
Segunda Neoplasia Primária , Neoplasias Testiculares , Humanos , Masculino , Movimento Celular , Mineração de Dados , Transporte Proteico
11.
12.
Int J Mol Sci ; 23(21)2022 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-36361553

RESUMO

Mutations in the apical Na-K-2Cl co-transporter, NKCC2, cause type I Bartter syndrome (BS1), a life-threatening kidney disease. We have previously demonstrated that the BS1 variant Y998X, which deprives NKCC2 from its highly conserved dileucine-like motifs, compromises co-transporter surface delivery through ER retention mechanisms. However, whether these hydrophobic motifs are sufficient for anterograde trafficking of NKCC2 remains to be determined. Interestingly, sequence analysis of NKCC2 C-terminus revealed the presence of consensus di-acidic (D/E-X-D/E) motifs, 949EEE951 and 1019DAELE1023, located upstream and downstream of BS1 mutation Y998X, respectively. Di-acidic codes are involved in ER export of proteins through interaction with COPII budding machinery. Importantly, whereas mutating 949EEE951 motif to 949AEA951 had no effect on NKCC2 processing, mutating 1019DAE1021 to 1019AAA1021 heavily impaired complex-glycosylation and cell surface expression of the cotransporter in HEK293 and OKP cells. Most importantly, triple mutation of D, E and E residues of 1019DAELE1023 to 1019AAALA1023 almost completely abolished NKCC2 complex-glycosylation, suggesting that this mutant failed to exit the ER. Cycloheximide chase analysis demonstrated that the absence of the terminally glycosylated form of 1019AAALA1023 was caused by defects in NKCC2 maturation. Accordingly, co-immunolocalization experiments revealed that 1019AAALA1023 was trapped in the ER. Finally, overexpression of a dominant negative mutant of Sar1-GTPase abolished NKCC2 maturation and cell surface expression, clearly indicating that NKCC2 export from the ER is COPII-dependent. Hence, our data indicate that in addition to the di-leucine like motifs, NKCC2 uses di-acidic exit codes for export from the ER through the COPII-dependent pathway. We propose that any naturally occurring mutation of NKCC2 interfering with this pathway could form the molecular basis of BS1.


Assuntos
Síndrome de Bartter , Simportadores , Humanos , Síndrome de Bartter/genética , Membrana Celular/metabolismo , Células HEK293 , Transporte Proteico , Simportadores/metabolismo
13.
Int J Mol Sci ; 23(21)2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36362297

RESUMO

Subcellular mRNA localization is an evolutionarily conserved mechanism to spatially and temporally drive local translation and, in turn, protein targeting. Hence, this mechanism achieves precise control of gene expression and establishes functional and structural networks during cell growth and development as well as during stimuli response. Since its discovery in ascidian eggs, mRNA localization has been extensively studied in animal and yeast cells. Although our knowledge of subcellular mRNA localization in plant cells lags considerably behind other biological systems, mRNA localization to the endoplasmic reticulum (ER) has also been well established since its discovery in cereal endosperm cells in the early 1990s. Storage protein mRNA targeting to distinct subdomains of the ER determines efficient accumulation of the corresponding proteins in different endosomal storage sites and, in turn, underlies storage organelle biogenesis in cereal grains. The targeting process requires the presence of RNA localization elements, also called zipcodes, and specific RNA-binding proteins that recognize and bind these zipcodes and recruit other factors to mediate active transport. Here, we review the current knowledge of the mechanisms and functions of mRNA localization to the ER in plant cells and address directions for future research.


Assuntos
Endosperma , Células Vegetais , Animais , Endosperma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Vegetais/metabolismo , Retículo Endoplasmático/metabolismo , Transporte Proteico , Grão Comestível/metabolismo
14.
Bioessays ; 44(12): e2200158, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36344475

RESUMO

Intercellular communication is an essential process in all multicellular organisms. During this process, molecules secreted by one cell will bind to a receptor on the cognate cell leading to the subsequent uptake of the receptor-ligand complex. Once inside, the cell then determines the fate of the receptor-ligand complex and any other proteins that were endocytosed together. Approximately 80% of endocytosed material is recycled back to the plasma membrane either directly or indirectly via the Golgi apparatus and the remaining 20% is delivered to the lysosome for degradation. Although most pathways have been identified, we still lack understanding on how specificity in sorting of recycling cargos into different pathways is achieved, and how the cell reaches high accuracy of these processes in the absence of clear sorting signals in the bulk of the client proteins. In this review, we will summarize our current understanding of the mechanism behind recycling cargo sorting and propose a model of differential affinities between cargo and cargo receptors/adaptors with regards to iterative sorting in endosomes.


Assuntos
Endocitose , Endossomos , Humanos , Ligantes , Endossomos/metabolismo , Transporte Proteico , Proteínas/metabolismo , Comunicação Celular
15.
Mol Biol Cell ; 33(14): ae4, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36399622

RESUMO

In 1956, referring to the emerging application of electron microscopy to the study of eukaryotic cells, Keith R. Porter wrote, "For those of us who are fortunate to be part of this new development, these are days of great interest and opportunity." Those early days left us a rich legacy of knowledge on the internal organization of eukaryotic cells that provides a framework for current research on cell structure and function. In this vein, my long-time quest has been to understand how proteins and organelles travel through the cytoplasm to reach their respective destinations within the cell. This research has led us to elucidate various mechanisms of protein sorting and organelle transport and how defects in these mechanisms cause human disease.


Assuntos
Organelas , Humanos , Organelas/metabolismo , Microscopia Eletrônica , Transporte Biológico/fisiologia , Citoplasma/metabolismo , Transporte Proteico
16.
Mol Neurodegener ; 17(1): 74, 2022 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-36397124

RESUMO

The family of VPS10p-Domain (D) receptors comprises five members named SorLA, Sortilin, SorCS1, SorCS2 and SorCS3. While their physiological roles remain incompletely resolved, they have been recognized for their signaling engagements and trafficking abilities, navigating a number of molecules between endosome, Golgi compartments, and the cell surface. Strikingly, recent studies connected all the VPS10p-D receptors to Alzheimer's disease (AD) development. In addition, they have been also associated with diseases comorbid with AD such as diabetes mellitus and major depressive disorder. This systematic review elaborates on genetic, functional, and mechanistic insights into how dysfunction in VPS10p-D receptors may contribute to AD etiology, AD onset diversity, and AD comorbidities. Starting with their functions in controlling cellular trafficking of amyloid precursor protein and the metabolism of the amyloid beta peptide, we present and exemplify how these receptors, despite being structurally similar, regulate various and distinct cellular events involved in AD. This includes a plethora of signaling crosstalks that impact on neuronal survival, neuronal wiring, neuronal polarity, and synaptic plasticity. Signaling activities of the VPS10p-D receptors are especially linked, but not limited to, the regulation of neuronal fitness and apoptosis via their physical interaction with pro- and mature neurotrophins and their receptors. By compiling the functional versatility of VPS10p-D receptors and their interactions with AD-related pathways, we aim to further propel the AD research towards VPS10p-D receptor family, knowledge that may lead to new diagnostic markers and therapeutic strategies for AD patients.


Assuntos
Doença de Alzheimer , Transtorno Depressivo Maior , Humanos , Peptídeos beta-Amiloides , Transporte Proteico/fisiologia , Fatores de Crescimento Neural
17.
Front Immunol ; 13: 1030409, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36439187

RESUMO

There are multiple regulatory layers that control intracellular trafficking and protein secretion, ranging from transcriptional to posttranslational mechanisms. Finely regulated trafficking and secretion is especially important for lymphocytes during activation and differentiation, as the quantity of secretory cargo increases once the activated cells start to produce and secrete large amounts of cytokines, cytotoxins, or antibodies. However, how the secretory machinery dynamically adapts its efficiency and specificity in general and specifically in lymphocytes remains incompletely understood. Here we present a systematic bioinformatics analysis to address RNA-based mechanisms that control intracellular trafficking and protein secretion during B-lymphocyte activation, and differentiation, with a focus on alternative splicing. Our in silico analyses suggest that alternative splicing has a substantial impact on the dynamic adaptation of intracellular traffic and protein secretion in different B cell subtypes, pointing to another regulatory layer to the control of lymphocyte function during activation and differentiation. Furthermore, we suggest that NERF/ELF2 controls the expression of some COPII-related genes in a cell type-specific manner. In addition, T cells and B cells appear to use different adaptive strategies to adjust their secretory machineries during the generation of effector and memory cells, with antibody secreting B cell specifically increasing the expression of components of the early secretory pathway. Together, our data provide hypotheses how cell type-specific regulation of the trafficking machinery during immune cell activation and differentiation is controlled that can now be tested in wet lab experiments.


Assuntos
Processamento Alternativo , Ativação Linfocitária , Ativação Linfocitária/genética , Transporte Proteico/fisiologia , Via Secretória , Linfócitos B
18.
Nat Commun ; 13(1): 6004, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36224181

RESUMO

Aberrant activation of EGFR due to overexpression or mutation is associated with poor prognosis in many types of tumors. Here we show that blocking the sorting system that directs EGFR to plasma membrane is a potent strategy to treat EGFR-dependent tumors. We find that EGFR palmitoylation by DHHC13 is critical for its plasma membrane localization and identify ARF6 as a key factor in this process. N-myristoylated ARF6 recognizes palmitoylated EGFR via lipid-lipid interaction, recruits the exocyst complex to promote EGFR budding from Golgi, and facilitates EGFR transporting to plasma membrane in a GTP-bound form. To evaluate the therapeutic potential of this sorting system, we design a cell-permeable peptide, N-myristoylated GKVL-TAT, and find it effectively disrupts plasma membrane localization of EGFR and significantly inhibits progression of EGFR-dependent tumors. Our findings shed lights on the underlying mechanism of how palmitoylation directs protein sorting and provide an potential strategy to manage EGFR-dependent tumors.


Assuntos
Fatores de Ribosilação do ADP , Neoplasias , Fatores de Ribosilação do ADP/metabolismo , Membrana Celular/metabolismo , Receptores ErbB/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Lipídeos , Neoplasias/metabolismo , Transporte Proteico
19.
Commun Biol ; 5(1): 1060, 2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36198903

RESUMO

Effective protein import from cytosol is critical for mitochondrial functions and metabolic regulation. We describe here the mammalian muscle-specific and systemic consequences to disrupted mitochondrial matrix protein import by targeted deletion of the mitochondrial HSP70 co-chaperone GRPEL1. Muscle-specific loss of GRPEL1 caused rapid muscle atrophy, accompanied by shut down of oxidative phosphorylation and mitochondrial fatty acid oxidation, and excessive triggering of proteotoxic stress responses. Transcriptome analysis identified new responders to mitochondrial protein import toxicity, such as the neurological disease-linked intermembrane space protein CHCHD10. Besides communication with ER and nucleus, we identified crosstalk of distressed mitochondria with peroxisomes, in particular the induction of peroxisomal Acyl-CoA oxidase 2 (ACOX2), which we propose as an ATF4-regulated peroxisomal marker of integrated stress response. Metabolic profiling indicated fatty acid enrichment in muscle, a shift in TCA cycle intermediates in serum and muscle, and dysregulated bile acids. Our results demonstrate the fundamental importance of GRPEL1 and provide a robust model for detecting mammalian inter-organellar and systemic responses to impaired mitochondrial matrix protein import and folding.


Assuntos
Ácidos Graxos , Músculo Esquelético , Animais , Ácidos e Sais Biliares/metabolismo , Ácidos Graxos/metabolismo , Mamíferos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Transporte Proteico
20.
J Cell Biol ; 221(12)2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36190447

RESUMO

Retromer-dependent endosomal recycling of membrane receptors requires Rab7, sorting nexin (SNX)-retromer, and factors that regulate endosomal actin organization. It is not fully understood how these factors cooperate to form endosomal subdomains for cargo retrieval and recycling. Here, we report that WDR91, a Rab7 effector, is the key factor that specifies the endosomal retrieval subdomain. Loss of WDR91 causes defective recycling of both intracellular and cell surface receptors. WDR91 interacts with SNXs through their PX domain, and with VPS35, thus promoting their interaction with Rab7. WDR91 also interacts with the WASH subunit FAM21. In WDR91-deficient cells, Rab7, SNX-retromer, and FAM21 fail to localize to endosomal subdomains, and endosomal actin organization is impaired. Re-expression of WDR91 enables Rab7, SNX-retromer, and FAM21 to concentrate at WDR91-specific endosomal subdomains, where retromer-mediated membrane tubulation and release occur. Thus, WDR91 coordinates Rab7 with SNX-retromer and WASH to establish the endosomal retrieval subdomains required for retromer-mediated endosomal recycling.


Assuntos
Proteínas de Transporte , Endossomos , Actinas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Endossomos/genética , Endossomos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Domínios Proteicos , Transporte Proteico/fisiologia , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo
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