Pub Date : 2021-12-01Epub Date: 2021-10-19DOI: 10.1111/tra.12816
Theodore L Steck, S M Ali Tabei, Yvonne Lange
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.
{"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":null,"pages":null},"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}
Pub 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
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.
虽然大多数单病毒科病毒只在宿主细胞质中复制,但其中许多病毒编码在细胞核和细胞质之间运输的蛋白质,这被认为在调节受感染宿主细胞的生物学方面具有辅助功能。其中,狂犬病毒的P3蛋白通过几种特定的核定位和核输出信号的活性定位到细胞核。P3的主要功能是逃避干扰素(IFN)介导的抗病毒反应,包括通过抑制干扰素激活的STAT1的dna结合。P3也定位于核仁和早幼粒细胞白血病(PML)核体,并与核仁蛋白和PML蛋白相互作用,表明其在核内具有多种作用。然而,P3核定位与致病性的关系尚不清楚。我们报道了致病性RABV毒株Nishigahara (Ni)和非致病性Ni衍生毒株Ni- ce中P3蛋白的核胞质定位存在显著差异,Ni- ce -P3的核积累存在缺陷。分子图谱表明,定位的改变源于协调效应,包括两个残基取代,这两个残基取代分别禁用核定位和增强核输出信号,共同促进核排斥。有趣的是,这似乎与对蛋白质构象或调节机制的影响有关,而不是直接修改已定义的运输信号序列。这些数据为在细胞质中复制的病毒的致病性中调节病毒蛋白的核运输的作用提供了新的见解。
{"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":null,"pages":null},"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}
Pub 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
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.
{"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":null,"pages":null},"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}
Pub Date : 2021-12-01Epub Date: 2021-10-05DOI: 10.1111/tra.12817
Sharanya Chatterjee, Ana Jeemin Choi, Gad Frankel
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.
内质网(ER)到高尔基体的运输是一个重要的和高度保守的细胞过程。转运机制的外壳蛋白复合物- ii (COPII)臂通过与COPII外壳的主要亚基Sec24的直接或间接相互作用,将大量的货物蛋白整合到囊泡中。大约三分之一的哺乳动物蛋白质依靠copii介导的分泌途径插入或分泌膜。有4个哺乳动物Sec24类似物和3个酵母Sec24类似物,有证据表明它们具有类似物特异性的货物相互作用基序。此外,个体相似物对货物蛋白上存在的分类基序子集的亲和力也有所不同。与蛋白质贩运的许多方面一样,我们对Sec24与货物的相互作用缺乏系统和彻底的了解。本系统综述的重点是目前对酵母和哺乳动物Sec24类似物及其内质网输出基序的货物结合的了解。分析表明,货物(和货物受体)的Sec24平行特异性范围从排他性平行依赖或偏好到部分冗余。我们还讨论了Sec24分泌系统如何被病毒(如VSV-G、乙型肝炎包膜蛋白)和细菌(如肠致病性大肠杆菌III型分泌系统效应物NleA/EspI)病原体劫持。
{"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":null,"pages":null},"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}
Pub 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
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.
{"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":null,"pages":null},"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}
Pub Date : 2021-11-01Epub Date: 2021-09-13DOI: 10.1111/tra.12813
Zoe Elizabeth West, Savannah Margaret Aitcheson, Annalese Barbara Trudy Semmler, Rachael Zoe Murray
The activity of the matrix metalloproteinase (MMP) MT1-MMP is strictly regulated by expression and cellular location. In macrophages LPS activation leads to the up-regulation of MT1-MMP and this need to be at the cell surface for them to degrade the dense extracellular matrix (ECM) components to create a path to migrate into injured and infected tissues. Fixed and live imaging shows newly made MT1-MMP is packaged into vesicles that traffic to and fuse with LBPA+ LAMP1+ late endosomes en route to the surface. The R-SNARE VAMP4, found on Golgi-derived vesicles that traffic to late endosomes, forms a trans-SNARE complex with the Q-SNARE complex Stx6/Stx7/Vti1b. The Stx6/Stx7/Vti1b complex has been shown to be up-regulated in lipopolysaccharide (LPS)-activated cells to increase trafficking of key cytokines through the classical pathway and now we show here it is up-regulation also plays a role in the late endosomal pathway of MT1-MMP trafficking. Depletion of any of the SNAREs in this complex reduces surface MT1-MMP and gelatin degradation. Conversely, overexpression of the Stx6/Stx7/Vti1b components increases surface MT1-MMP levels. This suggests that Stx6/Stx7/Vti1b is a key Q-SNARE complex in macrophages during an immune response and in partnership with VAMP4 it regulates transport of newly made MT1-MMP.
{"title":"The trans-SNARE complex VAMP4/Stx6/Stx7/Vti1b is a key regulator of Golgi to late endosome MT1-MMP transport in macrophages.","authors":"Zoe Elizabeth West, Savannah Margaret Aitcheson, Annalese Barbara Trudy Semmler, Rachael Zoe Murray","doi":"10.1111/tra.12813","DOIUrl":"https://doi.org/10.1111/tra.12813","url":null,"abstract":"<p><p>The activity of the matrix metalloproteinase (MMP) MT1-MMP is strictly regulated by expression and cellular location. In macrophages LPS activation leads to the up-regulation of MT1-MMP and this need to be at the cell surface for them to degrade the dense extracellular matrix (ECM) components to create a path to migrate into injured and infected tissues. Fixed and live imaging shows newly made MT1-MMP is packaged into vesicles that traffic to and fuse with LBPA<sup>+</sup> LAMP1<sup>+</sup> late endosomes en route to the surface. The R-SNARE VAMP4, found on Golgi-derived vesicles that traffic to late endosomes, forms a trans-SNARE complex with the Q-SNARE complex Stx6/Stx7/Vti1b. The Stx6/Stx7/Vti1b complex has been shown to be up-regulated in lipopolysaccharide (LPS)-activated cells to increase trafficking of key cytokines through the classical pathway and now we show here it is up-regulation also plays a role in the late endosomal pathway of MT1-MMP trafficking. Depletion of any of the SNAREs in this complex reduces surface MT1-MMP and gelatin degradation. Conversely, overexpression of the Stx6/Stx7/Vti1b components increases surface MT1-MMP levels. This suggests that Stx6/Stx7/Vti1b is a key Q-SNARE complex in macrophages during an immune response and in partnership with VAMP4 it regulates transport of newly made MT1-MMP.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2021-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/tra.12813","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39380588","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}
Pub Date : 2021-11-01Epub Date: 2021-09-23DOI: 10.1111/tra.12815
Katherine M Paine, Gabrielle B Ecclestone, Chris MacDonald
Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the uracil permease Fur4 in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a library of haploid deletion strains and identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multisubunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterized genes. Bioinformatic analysis of expression profiles from many strains lacking transcription factors required for efficient uracil-scavenging validated particular hits from the screen, in addition to implicating essential genes not tested in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorize factors implicated in uracil-scavenging.
{"title":"Fur4-mediated uracil-scavenging to screen for surface protein regulators.","authors":"Katherine M Paine, Gabrielle B Ecclestone, Chris MacDonald","doi":"10.1111/tra.12815","DOIUrl":"https://doi.org/10.1111/tra.12815","url":null,"abstract":"<p><p>Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the uracil permease Fur4 in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a library of haploid deletion strains and identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multisubunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterized genes. Bioinformatic analysis of expression profiles from many strains lacking transcription factors required for efficient uracil-scavenging validated particular hits from the screen, in addition to implicating essential genes not tested in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorize factors implicated in uracil-scavenging.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2021-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/tra.12815","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39397432","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}
Pub Date : 2021-10-01Epub Date: 2021-08-27DOI: 10.1111/tra.12811
Ashwani Sharma, G Aditya Kumar, Amitabha Chattopadhyay
Smith-Lemli-Opitz syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. It is characterized by accumulation of 7-dehydrocholesterol (the immediate biosynthetic precursor of cholesterol in the Kandutsch-Russell pathway) and an altered cholesterol to total sterol ratio. Because SLOS is associated with neurological malfunction, exploring the function and trafficking of neuronal receptors and their interaction with membrane lipids under these conditions assume significance. In this work, we generated a cellular model of SLOS in HEK-293 cells stably expressing the human serotonin1A receptor (an important neurotransmitter G-protein coupled receptor) using AY 9944, an inhibitor for the enzyme 3β-hydroxy-steroid-∆7 -reductase (7-DHCR). Using a quantitative flow cytometry based assay, we show that the plasma membrane population of serotonin1A receptors was considerably reduced under these conditions without any change in total cellular expression of the receptor. Interestingly, the receptors were trafficked to sterol-enriched LysoTracker positive compartments, which accumulated under these conditions. To the best of our knowledge, our results constitute one of the first reports demonstrating intracellular accumulation and misregulated traffic of a neurotransmitter GPCR in SLOS-like conditions. We believe these results assume relevance in our overall understanding of the molecular basis underlying the functional relevance of neurotransmitter receptors in SLOS.
{"title":"Late endosomal/lysosomal accumulation of a neurotransmitter receptor in a cellular model of Smith-Lemli-Opitz syndrome.","authors":"Ashwani Sharma, G Aditya Kumar, Amitabha Chattopadhyay","doi":"10.1111/tra.12811","DOIUrl":"https://doi.org/10.1111/tra.12811","url":null,"abstract":"<p><p>Smith-Lemli-Opitz syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. It is characterized by accumulation of 7-dehydrocholesterol (the immediate biosynthetic precursor of cholesterol in the Kandutsch-Russell pathway) and an altered cholesterol to total sterol ratio. Because SLOS is associated with neurological malfunction, exploring the function and trafficking of neuronal receptors and their interaction with membrane lipids under these conditions assume significance. In this work, we generated a cellular model of SLOS in HEK-293 cells stably expressing the human serotonin<sub>1A</sub> receptor (an important neurotransmitter G-protein coupled receptor) using AY 9944, an inhibitor for the enzyme 3β-hydroxy-steroid-∆<sup>7</sup> -reductase (7-DHCR). Using a quantitative flow cytometry based assay, we show that the plasma membrane population of serotonin<sub>1A</sub> receptors was considerably reduced under these conditions without any change in total cellular expression of the receptor. Interestingly, the receptors were trafficked to sterol-enriched LysoTracker positive compartments, which accumulated under these conditions. To the best of our knowledge, our results constitute one of the first reports demonstrating intracellular accumulation and misregulated traffic of a neurotransmitter GPCR in SLOS-like conditions. We believe these results assume relevance in our overall understanding of the molecular basis underlying the functional relevance of neurotransmitter receptors in SLOS.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2021-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/tra.12811","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39331487","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}
Claudia Metz, Claudia Oyanadel, Juan Jung, Claudio Retamal, Jorge Cancino, Jonathan Barra, Jaime Venegas, Guangwei Du, Andrea Soza, Alfonso González
Ligand-independent epidermal growth factor receptor (EGFR) endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid (PA) signaling toward the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and protein kinase A (PKA) activity. This PA/PDE4/PKA pathway is triggered with propranolol used to inhibit PA hydrolysis and induces clathrin-dependent and clathrin-independent endocytosis, followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this signaling pathway using biosensors of PA, cAMP, and PKA in live cells and then show that it activates p38 and ERK1/2 downstream the PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increases the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is probably because of EGFR phosphorylation, as detected in Th1046/1047 and Ser669 residues. The EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin, and Rab11, while a small proportion distributes to Alix-endosomes. A non-selective recycling arrest includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer.
{"title":"Phosphatidic acid-PKA signaling regulates p38 and ERK1/2 functions in ligand-independent EGFR endocytosis.","authors":"Claudia Metz, Claudia Oyanadel, Juan Jung, Claudio Retamal, Jorge Cancino, Jonathan Barra, Jaime Venegas, Guangwei Du, Andrea Soza, Alfonso González","doi":"10.1111/tra.12812","DOIUrl":"https://doi.org/10.1111/tra.12812","url":null,"abstract":"<p><p>Ligand-independent epidermal growth factor receptor (EGFR) endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid (PA) signaling toward the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and protein kinase A (PKA) activity. This PA/PDE4/PKA pathway is triggered with propranolol used to inhibit PA hydrolysis and induces clathrin-dependent and clathrin-independent endocytosis, followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this signaling pathway using biosensors of PA, cAMP, and PKA in live cells and then show that it activates p38 and ERK1/2 downstream the PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increases the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is probably because of EGFR phosphorylation, as detected in Th1046/1047 and Ser669 residues. The EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin, and Rab11, while a small proportion distributes to Alix-endosomes. A non-selective recycling arrest includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2021-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/tra.12812","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39342407","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}
Pub Date : 2021-10-01Epub Date: 2021-08-17DOI: 10.1111/tra.12810
Eric Chevet, Maria Antonietta De Matteis, Eeva-Liisa Eskelinen, Hesso Farhan
In this article we discuss implications of the recent discovery of glycoRNAs found to be present at the cell surface of mammalian cells which was reported by Flynn et al. Cell 2021.
{"title":"RNA, a new member in the glycan-club that gets exposed at the cell surface.","authors":"Eric Chevet, Maria Antonietta De Matteis, Eeva-Liisa Eskelinen, Hesso Farhan","doi":"10.1111/tra.12810","DOIUrl":"https://doi.org/10.1111/tra.12810","url":null,"abstract":"<p><p>In this article we discuss implications of the recent discovery of glycoRNAs found to be present at the cell surface of mammalian cells which was reported by Flynn et al. Cell 2021.</p>","PeriodicalId":23207,"journal":{"name":"Traffic","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2021-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/tra.12810","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39267488","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}