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1.
Blood Adv ; 6(17): 5198-5209, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36069828

RESUMO

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D'D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D'D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D'D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.


Assuntos
Complexo de Golgi , Fator de von Willebrand , Sítios de Ligação , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Domínios Proteicos , Fator de von Willebrand/metabolismo
2.
Sci Rep ; 12(1): 14975, 2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36056100

RESUMO

Retro-2 directly interacts with an ER exit site protein, Sec16A, inhibiting ER exit of a Golgi tSNARE, Syntaxin5, which results in rapid re-distribution of Syntaxin5 to the ER. Recently, it was shown that SARS-CoV-2 infection disrupts the Golgi apparatus within 6-12 h, while its replication was effectively inhibited by Retro-2 in cultured human lung cells. Yet, exactly how Retro-2 may influence ultrastructure of the Golgi apparatus have not been thoroughly investigated. In this study, we characterized the effect of Retro-2 treatment on ultrastructure of the Golgi apparatus using electron microscopy and EM tomography. Our initial results on protein secretion showed that Retro-2 treatment does not significantly influence secretion of either small or large cargos. Ultra-structural study of the Golgi, however, revealed rapid accumulation of COPI-like vesicular profiles in the perinuclear area and a partial disassembly of the Golgi stack under electron microscope within 3-5 h, suggesting altered Golgi organization in these cells. Retro-2 treatment in cells depleted of GRASP65/55, the two well-known Golgi structural proteins, induced complete and rapid disassembly of the Golgi into individual cisterna. Taken together, these results suggest that Retro-2 profoundly alters Golgi structure to a much greater extent than previously anticipated.


Assuntos
COVID-19 , Complexo de Golgi , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , SARS-CoV-2 , Proteínas de Transporte Vesicular/metabolismo
3.
J Cell Biol ; 221(11)2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36125415

RESUMO

Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both "early" and "recycling" endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved.


Assuntos
Endossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ubiquitina , Proteínas de Transporte Vesicular , Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Proteínas R-SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/metabolismo
4.
Cell Rep ; 40(9): 111282, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-36044848

RESUMO

The Golgi complex is the central sorting station of the eukaryotic secretory pathway. Traffic through the Golgi requires activation of Arf guanosine triphosphatases that orchestrate cargo sorting and vesicle formation by recruiting an array of effector proteins. Arf activation and Golgi membrane association is controlled by large guanine nucleotide exchange factors (GEFs) possessing multiple conserved regulatory domains. Here we present cryoelectron microscopy (cryoEM) structures of full-length Gea2, the yeast paralog of the human Arf-GEF GBF1, that reveal the organization of these regulatory domains and explain how Gea2 binds to the Golgi membrane surface. We find that the GEF domain adopts two different conformations compatible with different stages of the Arf activation reaction. The structure of a Gea2-Arf1 activation intermediate suggests that the movement of the GEF domain primes Arf1 for membrane insertion upon guanosine triphosphate binding. We propose that conformational switching of Gea2 during the nucleotide exchange reaction promotes membrane insertion of Arf1.


Assuntos
Fator 1 de Ribosilação do ADP , Complexo de Golgi , Fatores de Troca do Nucleotídeo Guanina , Proteínas de Saccharomyces cerevisiae , Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Microscopia Crioeletrônica , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Biochim Biophys Acta Mol Cell Res ; 1869(11): 119345, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36007678

RESUMO

Numerous Golgi-resident enzymes implicated in glycosylation are regulated by the conserved intramembrane protease SPPL3. SPPL3-catalyzed endoproteolysis separates Golgi enzymes from their membrane anchors, enabling subsequent release from the Golgi and secretion. Experimentally altered SPPL3 expression changes glycosylation patterns, yet the regulation of SPPL3-mediated Golgi enzyme cleavage is not understood and conflicting results regarding the subcellular localization of SPPL3 have been reported. Here, we used precise genome editing to generate isogenic cell lines expressing N- or C-terminally tagged SPPL3 from its endogenous locus. Using these cells, we conducted co-localization analyses of tagged endogenous SPPL3 and Golgi markers under steady-state conditions and upon treatment with drugs disrupting Golgi organization. Our data demonstrate that endogenous SPPL3 is Golgi-resident and found predominantly in the mid-Golgi. We find that endogenous SPPL3 co-localizes with its substrates but similarly with non-substrate type II proteins, demonstrating that in addition to co-localization in the Golgi other substrate-intrinsic properties govern SPPL3-mediated intramembrane proteolysis. Given the prevalence of SPPL3-mediated cleavage among Golgi-resident proteins our results have important implications for the regulation of SPPL3 and its role in the organization of the Golgi glycosylation machinery.


Assuntos
Ácido Aspártico Endopeptidases , Glicosiltransferases , Ácido Aspártico Endopeptidases/genética , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Complexo de Golgi/metabolismo
6.
EMBO J ; 41(17): e112181, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35929178

RESUMO

Li et al present the results of a proximity-interaction screen in mammalian cells for the effector proteins of 25 members of the Arf family of small GTPases. This study has generated an important resource for those working in several areas of cell biology and provided an initial characterisation of two new cellular roles for some of the least well studied members of this family, the regulation of PLD1 by ARL11/14 in phagocytosis, and the regulation of PI4KB by ARL5A/5B in the Golgi.


Assuntos
Fatores de Ribosilação do ADP , Complexo de Golgi , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Complexo de Golgi/metabolismo , Mamíferos
7.
Cell Rep ; 40(7): 111195, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35977480

RESUMO

ATG9A is a highly conserved membrane protein required for autophagy initiation. It is trafficked from the trans-Golgi network (TGN) to the phagophore to act as a membrane source for autophagosome expansion. Here, we show that ATG9A is not just a passenger protein in the TGN but rather works in concert with GRASP55, a stacking factor for Golgi structure, to organize Golgi dynamics and integrity. Upon heat stress, the E3 ubiquitin ligase MARCH9 is promoted to ubiquitinate ATG9A in the form of K63 conjugation, and the nondegradable ubiquitinated ATG9A disperses from the Golgi apparatus to the cytoplasm more intensely, accompanied by inhibiting GRASP55 oligomerization, further resulting in Golgi fragmentation. Knockout of ATG9A or MARCH9 largely prevents Golgi fragmentation and protects Golgi functions under heat and other Golgi stresses. Our results reveal a noncanonical function of ATG9A for Golgi dynamics and suggest the pathway for sensing Golgi stress via the MARCH9/ATG9A axis.


Assuntos
Autofagossomos , Complexo de Golgi , Autofagossomos/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico , Ubiquitina/metabolismo , Rede trans-Golgi/metabolismo
8.
Anal Biochem ; 655: 114846, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-35973625

RESUMO

Analysis of the full spectrum of secreted proteins in cell culture is complicated by leakage of intracellular proteins from damaged cells. To address this issue, we compared the abundance of individual proteins between the cell lysate and the conditioned medium, reasoning that secreted proteins should be relatively more abundant in the conditioned medium. Marked enrichment for signal-peptide-bearing proteins with increasing conditioned media to cell lysate ratio, as well loss of this signal following brefeldin A treatment, confirmed the sensitivity and specificity of this approach. The subset of proteins demonstrating increased conditioned media to cell lysate ratio in the presence of Brefeldin A identified candidates for unconventional secretion via a pathway independent of ER to Golgi trafficking.


Assuntos
Complexo de Golgi , Proteínas , Brefeldina A/metabolismo , Brefeldina A/farmacologia , Meios de Cultivo Condicionados/metabolismo , Complexo de Golgi/metabolismo , Proteínas/metabolismo
9.
Cell Calcium ; 106: 102634, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35917684

RESUMO

Amongst the superfamily of transient receptor potential (TRP) channels, TRPV5 and TRPV6 are specialized members that mediate Ca2+-selective transport across epithelial membranes. Intriguingly, fluorescent fusion proteins of TRPV5 or TRPV6 are hardly discernible within the plasma membrane of living cells. Instead, TRPV6 is mostly found in vesicular membrane compartments, indicating either a rapid degradation or cycling of channel-bearing vesicles between endomembrane compartments and the plasma membrane. In TRPV6-expressing cells, brefeldin A, a toxin that blocks the transit between the endoplasmic reticulum and the Golgi apparatus, caused a drop in [Ca2+]i with a half time in the range of 0.5-1 h. Upon wash-out of the toxin, the [Ca2+]i rose to a steady-state level within 2-3 h. Consistently, the synchronized forward trafficking of TRPV6VL-eGFP after brefeldin A wash-out led to a visible accumulation of the protein within the plasma membrane, as shown by confocal and total internal reflection microscopy. Analysis of the internalization route and differentiation of vesicle populations provided evidence for a clathrin-dependent internalization pathway. Most TRPV6VL-bearing vesicles co-stained with Rab5a, a marker protein for early endosomes. Fewer vesicles were co-localized with Rab7a (late endosomes) or with Rab11 (recycling endosomes). From these data, we propose that the lack of plasma membrane visibility of the channel results from a rapid internalization, which in addition to transcriptional regulation, adds a layer of functional channel regulation to modulate transepithelial Ca2+ transport.


Assuntos
Cálcio , Canais de Cátion TRPV , Brefeldina A/metabolismo , Brefeldina A/farmacologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Canais de Cátion TRPV/metabolismo
10.
Nat Commun ; 13(1): 4494, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35918380

RESUMO

Enzymes from pathogens often modulate host protein post-translational modifications (PTMs), facilitating survival and proliferation of pathogens. Shigella virulence factors IpaJ and IcsB induce proteolytic cleavage and lysine fatty acylation on host proteins, which cause Golgi stress and suppress innate immunity, respectively. However, it is unknown whether host enzymes could reverse such modifications introduced by pathogens' virulence factors to suppress pathogenesis. Herein, we report that SIRT2, a potent lysine defatty-acylase, is upregulated by the transcription factor CREB3 under Golgi stress induced by Shigella infection. SIRT2 in turn removes the lysine fatty acylation introduced by Shigella virulence factor IcsB to enhance host innate immunity. SIRT2 knockout mice are more susceptible to Shigella infection than wildtype mice, demonstrating the importance of SIRT2 to counteract Shigella infection.


Assuntos
Disenteria Bacilar , Shigella , Acilação , Animais , Disenteria Bacilar/metabolismo , Complexo de Golgi/metabolismo , Lisina/metabolismo , Camundongos , Sirtuína 2/genética , Sirtuína 2/metabolismo , Fatores de Virulência/metabolismo
11.
Nat Commun ; 13(1): 4685, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35948564

RESUMO

The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1.


Assuntos
Complexo de Golgi , Lisossomos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo de Golgi/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosforilação
12.
Bioessays ; 44(10): e2200064, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35986435

RESUMO

An entirely different mechanism and localization were recently proposed for the COPII coat complex, challenging its well-accepted function to select and concentrate cargo into small COPII-coated spherical transport vesicles. Instead, the COPII complex is suggested to form a dynamic yet stationary collar that forms a boundary between the ER and the ER export membrane domain. This membrane domain, the ER exit site (ERES), is the site of COPII-mediated sorting and concentration of transport competent proteins. Subsequently, the ERES is implicated to mature and bud to form a sizeable pleiomorphic transport carrier that translocate on microtubules to fuse with the Golgi apparatus. Despite this drastic mechanistic dogma shift, most of the underlying protein-protein and protein-membrane interactions remain unchanged. Here, we attempt to provide a detailed description of the newly proposed model of how ER to Golgi transport works by describing the role of several essential proteins of the transport machinery.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório , Complexo de Golgi , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico
13.
Int J Mol Sci ; 23(15)2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35955719

RESUMO

Ceramide transport protein (CERT) mediates ceramide transfer from the endoplasmic reticulum to the Golgi for sphingomyelin (SM) biosynthesis. CERT is inactivated by multiple phosphorylation at the serine-repeat motif (SRM), and mutations that impair the SRM phosphorylation are associated with a group of inherited intellectual disorders in humans. It has been suggested that the N-terminal phosphatidylinositol 4-monophosphate [PtdIns(4)P] binding domain and the C-terminal ceramide-transfer domain of CERT physically interfere with each other in the SRM phosphorylated state, thereby repressing the function of CERT; however, it remains unclear which regions in CERT are involved in the SRM phosphorylation-dependent repression of CERT. Here, we identified a previously uncharacterized cluster of lysine/arginine residues that were predicted to be located on the outer surface of a probable coiled-coil fold in CERT. Substitutions of the basic amino acids in the cluster with alanine released the SRM-dependent repression of CERT activities, i.e., the synthesis of SM, PtdIns(4)P-binding, vesicle-associated membrane protein-associated protein (VAP) binding, ceramide-transfer activity, and localization to the Golgi, although the effect on SM synthesis activity was only partially compromised by the alanine substitutions, which moderately destabilized the trimeric status of CERT. These results suggest that the basic amino acid cluster in the coiled-coil region is involved in the regulation of CERT function.


Assuntos
Proteínas de Transporte , Ceramidas , Alanina/metabolismo , Aminoácidos Básicos/metabolismo , Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Ceramidas/metabolismo , Complexo de Golgi/metabolismo , Humanos , Fosfatidilinositóis/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases , Serina/metabolismo
14.
J Biomed Sci ; 29(1): 56, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35927755

RESUMO

All cells in the changing tumor microenvironment (TME) need a class of checkpoints to regulate the balance among exocytosis, endocytosis, recycling and degradation. The vesicular trafficking and secretion pathways regulated by the small Rab GTPases and their effectors convey cell growth and migration signals and function as meditators of intercellular communication and molecular transfer. Recent advances suggest that Rab proteins govern conventional and unconventional vesicular secretion pathways by trafficking widely diverse cargoes and substrates in remodeling TME. The mechanisms underlying the regulation of conventional and unconventional vesicular secretion pathways, their action modes and impacts on the cancer and stromal cells have been the focus of much attention for the past two decades. In this review, we discuss the current understanding of vesicular secretion pathways in TME. We begin with an overview of the structure, regulation, substrate recognition and subcellular localization of vesicular secretion pathways. We then systematically discuss how the three fundamental vesicular secretion processes respond to extracellular cues in TME. These processes are the conventional protein secretion via the endoplasmic reticulum-Golgi apparatus route and two types of unconventional protein secretion via extracellular vesicles and secretory autophagy. The latest advances and future directions in vesicular secretion-involved interplays between tumor cells, stromal cell and host immunity are also described.


Assuntos
Via Secretória , Microambiente Tumoral , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico , Proteínas rab de Ligação ao GTP/metabolismo
15.
Sci Adv ; 8(31): eabm5578, 2022 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-35921411

RESUMO

Lysosomes are central organelles for cellular degradation and energy metabolism. Neuronal ceroid lipofuscinoses (NCLs) are a group of the most common neurodegenerative lysosomal storage disorders characterized by intracellular accumulation of ceroid in neurons. Mutations in KCTD7, a gene encoding an adaptor of the CUL3-RING E3 ubiquitin ligase (CRL3) complex, are categorized as a unique NCL subtype. However, the underlying mechanisms remain elusive. Here, we report various lysosomal and autophagic defects in KCTD7-deficient cells. Mechanistically, the CRL3-KCTD7 complex degrades CLN5, whereas patient-derived KCTD7 mutations disrupt the interaction between KCTD7-CUL3 or KCTD7-CLN5 and ultimately lead to excessive accumulation of CLN5. The accumulated CLN5 disrupts the interaction between CLN6/8 and lysosomal enzymes at the endoplasmic reticulum (ER), subsequently impairing ER-to-Golgi trafficking of lysosomal enzymes. Our findings reveal previously unrecognized roles of KCTD7-mediated CLN5 proteolysis in lysosomal homeostasis and demonstrate that KCTD7 and CLN5 are biochemically linked and function in a common neurodegenerative pathway.


Assuntos
Lipofuscinoses Ceroides Neuronais , Complexo de Golgi/metabolismo , Humanos , Hidrolases , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Canais de Potássio/metabolismo
16.
Int J Mol Sci ; 23(15)2022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35955785

RESUMO

Nucleotide sugars (NSs) serve as substrates for glycosylation reactions. The majority of these compounds are synthesized in the cytoplasm, whereas glycosylation occurs in the endoplasmic reticulum (ER) and Golgi lumens, where catalytic domains of glycosyltransferases (GTs) are located. Therefore, translocation of NS across the organelle membranes is a prerequisite. This process is thought to be mediated by a group of multi-transmembrane proteins from the SLC35 family, i.e., nucleotide sugar transporters (NSTs). Despite many years of research, some uncertainties/inconsistencies related with the mechanisms of NS transport and the substrate specificities of NSTs remain. Here we present a comprehensive review of the NS import into the mammalian Golgi, which consists of three major parts. In the first part, we provide a historical view of the experimental approaches used to study NS transport and evaluate the most important achievements. The second part summarizes various aspects of knowledge concerning NSTs, ranging from subcellular localization up to the pathologies related with their defective function. In the third part, we present the outcomes of our research performed using mammalian cell-based models and discuss its relevance in relation to the general context.


Assuntos
Complexo de Golgi , Nucleotídeos , Açúcares , Animais , Transporte Biológico , Glicosilação , Complexo de Golgi/metabolismo , Mamíferos/metabolismo , Nucleotídeos/metabolismo , Açúcares/metabolismo
18.
EMBO J ; 41(18): e110596, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35938214

RESUMO

Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER-to-Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER-to-Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER.


Assuntos
Mecanotransdução Celular , Proteínas Monoméricas de Ligação ao GTP , Transporte Biológico , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transporte Proteico/fisiologia
19.
J Biol Chem ; 298(8): 102219, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35780830

RESUMO

Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease-related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA-binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum-Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington's disease.


Assuntos
Doenças Neurodegenerativas , Animais , Autofagossomos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Lisossomos/metabolismo , Camundongos , Doenças Neurodegenerativas/metabolismo
20.
EMBO J ; 41(17): e110698, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35844135

RESUMO

The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle-structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phospholipase D1 (PLD1) preferentially interacts with two closely related but poorly studied Arf family GTPases, ARL11 and ARL14, showing that PLD1 is activated by ARL11/14 and may recruit these GTPases to membrane vesicles, and that PLD1 and ARL11 collaborate to promote macrophage phagocytosis. Moreover, ARL5A and ARL5B were found to interact with and recruit phosphatidylinositol 4-kinase beta (PI4KB) at trans-Golgi, thus promoting PI4KB's function in PI4P synthesis and protein secretion.


Assuntos
1-Fosfatidilinositol 4-Quinase , Fosfolipase D , GTP Fosfo-Hidrolases/metabolismo , Complexo de Golgi/metabolismo , Fosfolipase D/química , Fosfolipase D/genética , Fosfolipase D/metabolismo
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