TrafficPub Date : 2022-02-01DOI: 10.1111/tra.12828
Dalton Buysse, Matt West, Mitchell Leih, Greg Odorizzi
{"title":"Bro1 binds the Vps20 subunit of ESCRT-III and promotes ESCRT-III regulation by Doa4.","authors":"Dalton Buysse, Matt West, Mitchell Leih, Greg Odorizzi","doi":"10.1111/tra.12828","DOIUrl":"https://doi.org/10.1111/tra.12828","url":null,"abstract":"<p><p>The budding of intralumenal vesicles (ILVs) at endosomes requires membrane scission by the ESCRT-III complex. This step is negatively regulated in yeast by Doa4, the ubiquitin hydrolase that deubiquitinates transmembrane proteins sorted as cargoes into ILVs. Doa4 acts non-enzymatically to inhibit ESCRT-III membrane scission activity by directly binding the Snf7 subunit of ESCRT-III. This interaction inhibits the remodeling/disassembly of Snf7 polymers required for the ILV membrane scission reaction. Thus, Doa4 is thought to have a structural role that delays ILV budding while it also functions enzymatically to deubiquitinate ILV cargoes. In this study, we show that Doa4 binding to Snf7 in vivo is antagonized by another ESCRT-III subunit, Vps20. Doa4 is restricted from interacting with Snf7 in yeast expressing a mutant Vps20 allele that constitutively binds Doa4. This inhibitory effect of Vps20 is suppressed by overexpression of another ESCRT-III-associated protein, Bro1. We show that Bro1 binds directly to Vps20, suggesting that Bro1 has a central role in relieving the antagonistic relationship that Vps20 has toward Doa4.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":"23 2","pages":"109-119"},"PeriodicalIF":4.5,"publicationDate":"2022-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8792227/pdf/nihms-1764596.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10601954","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
TrafficPub Date : 2022-01-30DOI: 10.1111/tra.12833
Shreya Goyal, Verónica A Segarra, Nitika, Aaron M Stetcher, Andrew W Truman, Adam M Reitzel, Richard J Chi
{"title":"Vps501, a novel vacuolar SNX-BAR protein cooperates with the SEA complex to regulate TORC1 signaling.","authors":"Shreya Goyal, Verónica A Segarra, Nitika, Aaron M Stetcher, Andrew W Truman, Adam M Reitzel, Richard J Chi","doi":"10.1111/tra.12833","DOIUrl":"10.1111/tra.12833","url":null,"abstract":"<p><p>The sorting nexins (SNX), constitute a diverse family of molecules that play varied roles in membrane trafficking, cell signaling, membrane remodeling, organelle motility and autophagy. In particular, the SNX-BAR proteins, a SNX subfamily characterized by a C-terminal dimeric Bin/Amphiphysin/Rvs (BAR) lipid curvature domain and a conserved Phox-homology domain, are of great interest. In budding yeast, many SNX-BARs proteins have well-characterized endo-vacuolar trafficking roles. Phylogenetic analyses allowed us to identify an additional SNX-BAR protein, Vps501, with a novel endo-vacuolar role. We report that Vps501 uniquely localizes to the vacuolar membrane and has physical and genetic interactions with the SEA complex to regulate TORC1 inactivation. We found cells displayed a severe deficiency in starvation-induced/nonselective autophagy only when SEA complex subunits are ablated in combination with Vps501, indicating a cooperative role with the SEA complex during TORC1 signaling during autophagy induction. Additionally, we found the SEACIT complex becomes destabilized in vps501Δsea1Δ cells, which resulted in aberrant endosomal TORC1 activity and subsequent Atg13 hyperphosphorylation. We have also discovered that the vacuolar localization of Vps501 is dependent upon a direct interaction with Sea1 and a unique lipid binding specificity that is also required for its function. This article is protected by copyright. All rights reserved.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":" ","pages":""},"PeriodicalIF":4.5,"publicationDate":"2022-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9305297/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39874342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
TrafficPub Date : 2022-01-01DOI: 10.1111/tra.12826
Chung-Ling Lu, Steven Ortmeier, Jon Brudvig, Tamara Moretti, Jacob Cain, Simeon A Boyadjiev, Jill M Weimer, Jinoh Kim
{"title":"Collagen has a unique SEC24 preference for efficient export from the endoplasmic reticulum.","authors":"Chung-Ling Lu, Steven Ortmeier, Jon Brudvig, Tamara Moretti, Jacob Cain, Simeon A Boyadjiev, Jill M Weimer, Jinoh Kim","doi":"10.1111/tra.12826","DOIUrl":"https://doi.org/10.1111/tra.12826","url":null,"abstract":"<p><p>SEC24 is mainly involved in cargo sorting during COPII vesicle assembly. There are four SEC24 paralogs (A-D) in vertebrates, which are classified into two subgroups (SEC24A/B and SEC24C/D). Pathological mutations in SEC24D cause osteogenesis imperfecta with craniofacial dysplasia in humans. sec24d mutant fish also recapitulate the phenotypes. Consistent with the skeletal phenotypes, the secretion of collagen was severely defective in mutant fish, emphasizing the importance of SEC24D in collagen secretion. However, SEC24D patient-derived fibroblasts show only a mild secretion phenotype, suggesting tissue-specificity in the secretion process. Using Sec24d KO mice and cultured cells, we show that SEC24A and SEC24B also contribute to endoplasmic reticulum (ER) export of procollagen. In contrast, fibronectin 1 requires either SEC24C or SEC24D for ER export. On the basis of our results, we propose that procollagen interacts with multiple SEC24 paralogs for efficient export from the ER, and that this is the basis for tissue-specific phenotypes resulting from SEC24 paralog deficiency.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":"23 1","pages":"81-93"},"PeriodicalIF":4.5,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8692420/pdf/nihms-1755513.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10806062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
TrafficPub Date : 2021-12-01Epub Date: 2021-10-03DOI: 10.1111/tra.12820
Bo Meng, Pedro P Vallejo Ramirez, Katharina M Scherer, Ezra Bruggeman, Julia C Kenyon, Clemens F Kaminski, Andrew M Lever
{"title":"EAP45 association with budding HIV-1: Kinetics and domain requirements.","authors":"Bo Meng, Pedro P Vallejo Ramirez, Katharina M Scherer, Ezra Bruggeman, Julia C Kenyon, Clemens F Kaminski, Andrew M Lever","doi":"10.1111/tra.12820","DOIUrl":"https://doi.org/10.1111/tra.12820","url":null,"abstract":"<p><p>A number of viruses including HIV use the ESCRT system to bud from the infected cell. We have previously confirmed biochemically that ESCRT-II is involved in this process in HIV-1 and have defined the molecular domains that are important for this. Here, using SNAP-tag fluorescent labelling and both fixed and live cell imaging we show that the ESCRT-II component EAP45 colocalises with the HIV protein Gag at the plasma membrane in a temporal and quantitative manner, similar to that previously shown for ALIX and Gag. We show evidence that a proportion of EAP45 may be packaged within virions, and we confirm the importance of the N terminus of EAP45 and specifically the H0 domain in this process. By contrast, the Glue domain of EAP45 is more critical for recruitment during cytokinesis, emphasising that viruses have ways of recruiting cellular components that may be distinct from those used by some cellular processes. This raises the prospect of selective interference with the pathway to inhibit viral function while leaving cellular functions relatively unperturbed.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":"22 12","pages":"439-453"},"PeriodicalIF":4.5,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39487518","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
TrafficPub Date : 2021-12-01Epub Date: 2021-09-27DOI: 10.1111/tra.12818
Andrew Samy, Noriko Yamano-Adachi, Yuichi Koga, Takeshi Omasa
{"title":"Secretion of a low-molecular-weight species of endogenous GRP94 devoid of the KDEL motif during endoplasmic reticulum stress in Chinese hamster ovary cells.","authors":"Andrew Samy, Noriko Yamano-Adachi, Yuichi Koga, Takeshi Omasa","doi":"10.1111/tra.12818","DOIUrl":"https://doi.org/10.1111/tra.12818","url":null,"abstract":"<p><p>GRP94 (glucose-regulated protein 94) is a well-studied chaperone with a lysine, aspartic acid, glutamic acid and leucine (KDEL) motif at its C-terminal, which is responsible for GRP94 localization in the endoplasmic reticulum (ER). GRP94 is upregulated during ER stress to help fold unfolded proteins or direct proteins to ER-associated degradation. In a previous study, engineered GRP94 without the KDEL motif stimulated a powerful immune response in vaccine cells. In this report, we show that endogenous GRP94 is naturally secreted into the medium in a truncated form that lacks the KDEL motif in Chinese hamster ovary cells. The secretion of the truncated form of GRP94 was stimulated by the induction of ER stress. These truncations prevent GRP94 recognition by KDEL receptors and retention inside the cell. This study sheds light on a potential trafficking phenomenon during the unfolded protein response that may help understand the functional role of GRP94 as a trafficking molecule.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":"22 12","pages":"425-438"},"PeriodicalIF":4.5,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/5d/10/TRA-22-425.PMC9293085.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39428718","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
TrafficPub Date : 2021-12-01Epub Date: 2021-10-19DOI: 10.1111/tra.12816
Theodore L Steck, S M Ali Tabei, Yvonne Lange
{"title":"A basic model for cell cholesterol homeostasis.","authors":"Theodore L Steck, S M Ali Tabei, Yvonne Lange","doi":"10.1111/tra.12816","DOIUrl":"https://doi.org/10.1111/tra.12816","url":null,"abstract":"<p><p>Cells manage their cholesterol by negative feedback using a battery of sterol-responsive proteins. How these activities are coordinated so as to specify the abundance and distribution of the sterol is unclear. We present a simple mathematical model that addresses this question. It assumes that almost all of the cholesterol is associated with phospholipids in stoichiometric complexes. A small fraction of the sterol is uncomplexed and thermodynamically active. It equilibrates among the organelles, setting their sterol level according to the affinity of their phospholipids. The activity of the homeostatic proteins in the cytoplasmic membranes is then set by their fractional saturation with uncomplexed cholesterol in competition with the phospholipids. The high-affinity phospholipids in the plasma membrane (PM) are filled to near stoichiometric equivalence, giving it most of the cell sterol. Notably, the affinity of the phospholipids in the endomembranes (EMs) is lower by orders of magnitude than that of the phospholipids in the PM. Thus, the small amount of sterol in the EMs rests far below stoichiometric capacity. Simulations match a variety of experimental data. The model captures the essence of cell cholesterol homeostasis, makes coherent a diverse set of experimental findings, provides a surprising prediction and suggests new experiments.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":"22 12","pages":"471-481"},"PeriodicalIF":4.5,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/tra.12816","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39420977","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
TrafficPub Date : 2021-12-01Epub Date: 2021-10-05DOI: 10.1111/tra.12819
Carlos O Oueslati Morales, Attila Ignácz, Norbert Bencsik, Zsofia Sziber, Anikó Erika Rátkai, Wolfgang S Lieb, Stephan A Eisler, Attila Szűcs, Katalin Schlett, Angelika Hausser
{"title":"Protein kinase D promotes activity-dependent AMPA receptor endocytosis in hippocampal neurons.","authors":"Carlos O Oueslati Morales, Attila Ignácz, Norbert Bencsik, Zsofia Sziber, Anikó Erika Rátkai, Wolfgang S Lieb, Stephan A Eisler, Attila Szűcs, Katalin Schlett, Angelika Hausser","doi":"10.1111/tra.12819","DOIUrl":"https://doi.org/10.1111/tra.12819","url":null,"abstract":"<p><p>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the brain. The continuous trafficking of AMPARs into and out of synapses is a core feature of synaptic plasticity, which is considered as the cellular basis of learning and memory. The molecular mechanisms underlying the postsynaptic AMPAR trafficking, however, are still not fully understood. In this work, we demonstrate that the protein kinase D (PKD) family promotes basal and activity-induced AMPAR endocytosis in primary hippocampal neurons. Pharmacological inhibition of PKD increased synaptic levels of GluA1-containing AMPARs, slowed down their endocytic trafficking and increased neuronal network activity. By contrast, ectopic expression of constitutive active PKD decreased the synaptic level of AMPARs, while increasing their colocalization with early endosomes. Our results thus establish an important role for PKD in the regulation of postsynaptic AMPAR trafficking during synaptic plasticity.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":"22 12","pages":"454-470"},"PeriodicalIF":4.5,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39451404","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
TrafficPub Date : 2021-12-01Epub Date: 2021-10-19DOI: 10.1111/tra.12821
Aaron M Brice, Ericka Watts, Bevan Hirst, David A Jans, Naoto Ito, Gregory W Moseley
{"title":"Implication of the nuclear trafficking of rabies virus P3 protein in viral pathogenicity.","authors":"Aaron M Brice, Ericka Watts, Bevan Hirst, David A Jans, Naoto Ito, Gregory W Moseley","doi":"10.1111/tra.12821","DOIUrl":"https://doi.org/10.1111/tra.12821","url":null,"abstract":"<p><p>Although the majority of viruses of the family Mononegvirales replicate exclusively in the host cell cytoplasm, many of these viruses encode proteins that traffic between the nucleus and cytoplasm, which is believed to enable accessory functions in modulating the biology of the infected host cell. Among these, the P3 protein of rabies virus localizes to the nucleus through the activity of several specific nuclear localization and nuclear export signals. The major defined functions of P3 are in evasion of interferon (IFN)-mediated antiviral responses, including through inhibition of DNA-binding by IFN-activated STAT1. P3 also localizes to nucleoli and promyelocytic leukemia (PML) nuclear bodies, and interacts with nucleolin and PML protein, indicative of several intranuclear roles. The relationship of P3 nuclear localization with pathogenicity, however, is unresolved. We report that nucleocytoplasmic localization of P3 proteins from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derived strain, Ni-CE, differs significantly, with nuclear accumulation defective for Ni-CE-P3. Molecular mapping indicates that altered localization derives from a coordinated effect, including two residue substitutions that independently disable nuclear localization and augment nuclear export signals, collectively promoting nuclear exclusion. Intriguingly, this appears to relate to effects on protein conformation or regulatory mechanisms, rather than direct modification of defined trafficking signal sequences. These data provide new insights into the role of regulated nuclear trafficking of a viral protein in the pathogenicity of a virus that replicates in the cytoplasm.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":"22 12","pages":"482-489"},"PeriodicalIF":4.5,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39521058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
TrafficPub Date : 2021-12-01Epub Date: 2021-10-05DOI: 10.1111/tra.12817
Sharanya Chatterjee, Ana Jeemin Choi, Gad Frankel
{"title":"A systematic review of Sec24 cargo interactome.","authors":"Sharanya Chatterjee, Ana Jeemin Choi, Gad Frankel","doi":"10.1111/tra.12817","DOIUrl":"https://doi.org/10.1111/tra.12817","url":null,"abstract":"<p><p>Endoplasmic reticulum (ER)-to-Golgi trafficking is an essential and highly conserved cellular process. The coat protein complex-II (COPII) arm of the trafficking machinery incorporates a wide array of cargo proteins into vesicles through direct or indirect interactions with Sec24, the principal subunit of the COPII coat. Approximately one-third of all mammalian proteins rely on the COPII-mediated secretory pathway for membrane insertion or secretion. There are four mammalian Sec24 paralogs and three yeast Sec24 paralogs with emerging evidence of paralog-specific cargo interaction motifs. Furthermore, individual paralogs also differ in their affinity for a subset of sorting motifs present on cargo proteins. As with many aspects of protein trafficking, we lack a systematic and thorough understanding of the interaction of Sec24 with cargoes. This systematic review focuses on the current knowledge of cargo binding to both yeast and mammalian Sec24 paralogs and their ER export motifs. The analyses show that Sec24 paralog specificity of cargo (and cargo receptors) range from exclusive paralog dependence or preference to partial redundancy. We also discuss how the Sec24 secretion system is hijacked by viral (eg, VSV-G, Hepatitis B envelope protein) and bacterial (eg, the enteropathogenic Escherichia coli type III secretion system effector NleA/EspI) pathogens.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":"22 12","pages":"412-424"},"PeriodicalIF":4.5,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/tra.12817","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39426871","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
TrafficPub Date : 2021-11-01Epub Date: 2021-09-27DOI: 10.1111/tra.12814
Shikha T Ramesh, Kolaparamba V Navyasree, Sneha Sah, Anjitha B Ashok, Nishada Qathoon, Suryasikha Mohanty, Rajeeb K Swain, Perunthottathu K Umasankar
{"title":"BMP2K phosphorylates AP-2 and regulates clathrin-mediated endocytosis.","authors":"Shikha T Ramesh, Kolaparamba V Navyasree, Sneha Sah, Anjitha B Ashok, Nishada Qathoon, Suryasikha Mohanty, Rajeeb K Swain, Perunthottathu K Umasankar","doi":"10.1111/tra.12814","DOIUrl":"https://doi.org/10.1111/tra.12814","url":null,"abstract":"<p><p>Phosphorylation of the central adaptor protein complex, AP-2 is pivotal for clathrin-mediated endocytosis (CME). Here, we uncover the role of an uncharacterized kinase (BMP-2 inducible kinase-BMP2K) in AP-2 phosphorylation. We demonstrate that BMP2K can phosphorylate AP-2 in vitro and in vivo. Functional impairment of BMP2K impedes AP-2 phosphorylation leading to defects in clathrin-coated pit (CCP) morphology and cargo internalization. BMP2K engages AP-2 via its extended C-terminus and this interaction is important for its CCP localization and function. Notably, endogenous BMP2K levels decline upon functional impairment of AP-2 indicating AP-2 dependent BMP2K stabilization in cells. Further, functional inactivation of BMP2K in zebrafish embryos yields gastrulation phenotypes which mirror AP-2 loss-of-function suggesting physiological relevance of BMP2K in vertebrates. Together, our findings propose involvement of a novel kinase in AP-2 phosphorylation and in the operation of CME.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":"22 11","pages":"377-396"},"PeriodicalIF":4.5,"publicationDate":"2021-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/tra.12814","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39383772","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}