Pub Date : 2014-05-01eCollection Date: 2014-06-01DOI: 10.4161/21592780.2014.943602
Richard A Kahn
Concepts or models of biological processes shape how we think about them, discuss them, and design experiments to test aspects of them. Because of the importance of our models of cell signaling by regulatory GTPases and the desire to extend those models to related signaling modules, I have throughout my career been fascinated by the similarities and differences between the modeling of heterotrimeric G protein and monomeric RAS superfamily GTPases. Recent discussions with colleagues led me to conclude that there is a growing divergence in how researchers model the activation and signaling processes of monomeric and trimeric GTPases and also a surprising lack of consensus within each camp. This series of articles arose in response to these discussions and is intended to spark new ones.
{"title":"Is the model of signal amplification by GPCRs/GEFs activating multiple GTPases relevant to a broad spectrum of heterotrimeric and RAS superfamily GTPases?","authors":"Richard A Kahn","doi":"10.4161/21592780.2014.943602","DOIUrl":"https://doi.org/10.4161/21592780.2014.943602","url":null,"abstract":"<p><p>Concepts or models of biological processes shape how we think about them, discuss them, and design experiments to test aspects of them. Because of the importance of our models of cell signaling by regulatory GTPases and the desire to extend those models to related signaling modules, I have throughout my career been fascinated by the similarities and differences between the modeling of heterotrimeric G protein and monomeric RAS superfamily GTPases. Recent discussions with colleagues led me to conclude that there is a growing divergence in how researchers model the activation and signaling processes of monomeric and trimeric GTPases and also a surprising lack of consensus within each camp. This series of articles arose in response to these discussions and is intended to spark new ones.</p>","PeriodicalId":72547,"journal":{"name":"Cellular logistics","volume":"4 2","pages":"e943602"},"PeriodicalIF":0.0,"publicationDate":"2014-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4161/21592780.2014.943602","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32993826","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-05-01eCollection Date: 2014-04-01DOI: 10.4161/21592780.2014.943618
Konstadinos Moissoglu, Martin Alexander Schwartz
Rho family GTPases control almost every aspect of cell physiology and, since their discovery, a wealth of knowledge has accumulated about their biochemical regulation and function. However, each Rho GTPase distributes between multiple cellular compartments, even within the same cell, where they are controlled by multiple regulators and signal to multiple effectors. Thus, major questions about spatial and temporal aspects of regulation remain unanswered. In particular, what are the nano-scale dynamics for their activation, membrane targeting, diffusion, effector activation and GTPase inactivation? How do these mechanisms differ in the different cellular compartments where Rho GTPases function? Addressing these complex aspects of Rho GTPase biology will significantly advance our understanding of the spatial and temporal control of cellular functions.
{"title":"Spatial and temporal control of Rho GTPase functions.","authors":"Konstadinos Moissoglu, Martin Alexander Schwartz","doi":"10.4161/21592780.2014.943618","DOIUrl":"https://doi.org/10.4161/21592780.2014.943618","url":null,"abstract":"<p><p>Rho family GTPases control almost every aspect of cell physiology and, since their discovery, a wealth of knowledge has accumulated about their biochemical regulation and function. However, each Rho GTPase distributes between multiple cellular compartments, even within the same cell, where they are controlled by multiple regulators and signal to multiple effectors. Thus, major questions about spatial and temporal aspects of regulation remain unanswered. In particular, what are the nano-scale dynamics for their activation, membrane targeting, diffusion, effector activation and GTPase inactivation? How do these mechanisms differ in the different cellular compartments where Rho GTPases function? Addressing these complex aspects of Rho GTPase biology will significantly advance our understanding of the spatial and temporal control of cellular functions.</p>","PeriodicalId":72547,"journal":{"name":"Cellular logistics","volume":"4 2","pages":"e943618"},"PeriodicalIF":0.0,"publicationDate":"2014-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4161/21592780.2014.943618","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32993830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-05-01eCollection Date: 2014-04-01DOI: 10.4161/21592780.2014.943616
Catherine L Jackson
Members of the Arf family of small GTP-binding proteins, or GTPases, are activated by guanine nucleotide exchange factors (GEFs) that catalyze GDP release from their substrate Arf, allowing GTP to bind. In the secretory pathway, Arf1 is first activated by GBF1 at the cis-Golgi, then by BIG1 and BIG2 at the trans-Golgi and trans-Golgi network (TGN). Upon activation, Arf1-GTP interacts with effectors such as coat complexes, and is able to recruit different coat complexes to different membrane sites in cells. The COPI coat is primarily recruited to cis-Golgi membranes, whereas other coats, such as AP-1/clathrin, and GGA/clathrin, are recruited to the trans-Golgi and the TGN. Although Arf1-GTP is required for stable association of these various coats to membranes, and is sufficient in vitro, other molecules, such as vesicle cargo and coat receptors on the membrane, contribute to specificity of coat recruitment in cells. Another mechanism to achieve specificity is interaction of effectors such as coats with the GEF itself, which would increase the concentration of a given coat in proximity to the site where Arf is activated, thus favoring its recruitment. This interaction between a GEF and an effector could also provide a mechanism for spatial organization of vesicle budding sites, similar to that described for Cdc42-mediated establishment of polarity sites such as the emerging bud in yeast. Another factor affecting the amount of freely diffusible Arf1-GTP in membranes is the GEF(s) themselves acting as effectors. Sec7p, the yeast homolog of mammalian BIG1 and BIG2, and Arno/cytohesin 2, a PM-localized Arf1 GEF, both bind to Arf1-GTP. This binding to the products of the exchange reaction establishes a positive feedback loop for activation.
{"title":"GEF-effector interactions.","authors":"Catherine L Jackson","doi":"10.4161/21592780.2014.943616","DOIUrl":"https://doi.org/10.4161/21592780.2014.943616","url":null,"abstract":"<p><p>Members of the Arf family of small GTP-binding proteins, or GTPases, are activated by guanine nucleotide exchange factors (GEFs) that catalyze GDP release from their substrate Arf, allowing GTP to bind. In the secretory pathway, Arf1 is first activated by GBF1 at the <i>cis</i>-Golgi, then by BIG1 and BIG2 at the <i>trans</i>-Golgi and <i>trans</i>-Golgi network (TGN). Upon activation, Arf1-GTP interacts with effectors such as coat complexes, and is able to recruit different coat complexes to different membrane sites in cells. The COPI coat is primarily recruited to <i>cis</i>-Golgi membranes, whereas other coats, such as AP-1/clathrin, and GGA/clathrin, are recruited to the <i>trans</i>-Golgi and the TGN. Although Arf1-GTP is required for stable association of these various coats to membranes, and is sufficient <i>in vitro</i>, other molecules, such as vesicle cargo and coat receptors on the membrane, contribute to specificity of coat recruitment in cells. Another mechanism to achieve specificity is interaction of effectors such as coats with the GEF itself, which would increase the concentration of a given coat in proximity to the site where Arf is activated, thus favoring its recruitment. This interaction between a GEF and an effector could also provide a mechanism for spatial organization of vesicle budding sites, similar to that described for Cdc42-mediated establishment of polarity sites such as the emerging bud in yeast. Another factor affecting the amount of freely diffusible Arf1-GTP in membranes is the GEF(s) themselves acting as effectors. Sec7p, the yeast homolog of mammalian BIG1 and BIG2, and Arno/cytohesin 2, a PM-localized Arf1 GEF, both bind to Arf1-GTP. This binding to the products of the exchange reaction establishes a positive feedback loop for activation.</p>","PeriodicalId":72547,"journal":{"name":"Cellular logistics","volume":"4 2","pages":"e943616"},"PeriodicalIF":0.0,"publicationDate":"2014-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4161/21592780.2014.943616","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32993827","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-04-03eCollection Date: 2014-01-01DOI: 10.4161/cl.28680
Nicholas W Baetz, James R Goldenring
The Rab11 GTPases and Rab11 family-interacting proteins (Rab11-FIPs) define integrated yet distinct compartments within the slow recycling pathway. The lipid content of these compartments is less well understood, although past studies have indicated phosphatidylserine (PS) is an integral component of recycling membranes. We sought to identify key differences in the presence of PS within Rab and Rab11-FIP containing membranes. We used live cell fluorescence microscopy and structured illumination microscopy to determine whether the previously published LactC2 probe for PS displays differential patterns of overlap with various Rab GTPases and Rab11-FIPs. Selective overlap was observed between the LactC2 probe and Rab GTPases when co-expressed in HeLa cells. Rab11-FIP1 proteins consistently overlapped with LactC2 along peripheral and pericentriolar compartments. The specificity of Rab11-FIP1 association with LactC2 was further confirmed by demonstrating that additional Rab11-FIPs (FIP2, FIP3, and FIP5) exhibited selective association with LactC2 containing compartments. Live cell dual expression studies of Rab11-FIPs with LactC2 indicated that PS is enriched along tubular compartments of the Rab11a-dependent recycling system. Additionally, we found that the removal of C2 domains from the Rab11-FIPs induced an accumulation of LactC2 probe in the pericentriolar region, suggesting that inhibition of trafficking through the recycling system can influence the distribution of PS within cells. Finally, we confirmed these findings using structured illumination microscopy suggesting that the overlapping fluorescent signals were on the same membranes. These results suggest distinct associations of Rab GTPases and Rab11-FIPs with PS-containing recycling system membrane domains.
{"title":"Distinct patterns of phosphatidylserine localization within the Rab11a-containing recycling system.","authors":"Nicholas W Baetz, James R Goldenring","doi":"10.4161/cl.28680","DOIUrl":"10.4161/cl.28680","url":null,"abstract":"<p><p>The Rab11 GTPases and Rab11 family-interacting proteins (Rab11-FIPs) define integrated yet distinct compartments within the slow recycling pathway. The lipid content of these compartments is less well understood, although past studies have indicated phosphatidylserine (PS) is an integral component of recycling membranes. We sought to identify key differences in the presence of PS within Rab and Rab11-FIP containing membranes. We used live cell fluorescence microscopy and structured illumination microscopy to determine whether the previously published LactC2 probe for PS displays differential patterns of overlap with various Rab GTPases and Rab11-FIPs. Selective overlap was observed between the LactC2 probe and Rab GTPases when co-expressed in HeLa cells. Rab11-FIP1 proteins consistently overlapped with LactC2 along peripheral and pericentriolar compartments. The specificity of Rab11-FIP1 association with LactC2 was further confirmed by demonstrating that additional Rab11-FIPs (FIP2, FIP3, and FIP5) exhibited selective association with LactC2 containing compartments. Live cell dual expression studies of Rab11-FIPs with LactC2 indicated that PS is enriched along tubular compartments of the Rab11a-dependent recycling system. Additionally, we found that the removal of C2 domains from the Rab11-FIPs induced an accumulation of LactC2 probe in the pericentriolar region, suggesting that inhibition of trafficking through the recycling system can influence the distribution of PS within cells. Finally, we confirmed these findings using structured illumination microscopy suggesting that the overlapping fluorescent signals were on the same membranes. These results suggest distinct associations of Rab GTPases and Rab11-FIPs with PS-containing recycling system membrane domains.</p>","PeriodicalId":72547,"journal":{"name":"Cellular logistics","volume":"4 ","pages":"e28680"},"PeriodicalIF":0.0,"publicationDate":"2014-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/52/8e/cl-4-e28680.PMC4156484.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32659380","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-03-18eCollection Date: 2014-01-01DOI: 10.4161/cl.28461
Shunsuke Kon, Nobuhide Kobayashi, Masanobu Satake
Ligand-stimulated receptor tyrosine kinases (RTKs) are phosphorylated/ubiquitinated, endocytosed and transported to the lysosomes via endosomes/multivesicular bodies, resulting in the attenuation of signal transmission. If this physiological mechanism of RTK signal downregulation is perturbed, signal transduction persists and may contribute to cellular transformation. This article presents several such examples. In some cases, endocytosis is impaired, and the activated RTK remains on the plasma membrane. In other cases, the activated RTK is endocytosed into endosomes/multivesicular bodies, but not subsequently sorted to the lysosomes for degradation. The latter cases indicate that even endocytosed RTKs can transmit signals. Transport of RTKs is accomplished via the formation and movement of membrane vesicles. Blockage or delay of endocytosis/trafficking can be caused by genetic alterations in the RTK itself or by mutations in CBL, Arf GAPs, or other components involved in internalization and vesicle transport. A survey of the literature indicates that, in some cases, even RTKs synthesized de novo can initiate signaling at the endoplasmic reticulum/Golgi before reaching the plasma membrane. The spectrum of molecules targeted by the signal is likely to be different between cell surface- and endoplasmic reticulum/Golgi-localized RTKs.
{"title":"Altered trafficking of mutated growth factor receptors and their associated molecules: implication for human cancers.","authors":"Shunsuke Kon, Nobuhide Kobayashi, Masanobu Satake","doi":"10.4161/cl.28461","DOIUrl":"https://doi.org/10.4161/cl.28461","url":null,"abstract":"<p><p>Ligand-stimulated receptor tyrosine kinases (RTKs) are phosphorylated/ubiquitinated, endocytosed and transported to the lysosomes via endosomes/multivesicular bodies, resulting in the attenuation of signal transmission. If this physiological mechanism of RTK signal downregulation is perturbed, signal transduction persists and may contribute to cellular transformation. This article presents several such examples. In some cases, endocytosis is impaired, and the activated RTK remains on the plasma membrane. In other cases, the activated RTK is endocytosed into endosomes/multivesicular bodies, but not subsequently sorted to the lysosomes for degradation. The latter cases indicate that even endocytosed RTKs can transmit signals. Transport of RTKs is accomplished via the formation and movement of membrane vesicles. Blockage or delay of endocytosis/trafficking can be caused by genetic alterations in the RTK itself or by mutations in CBL, Arf GAPs, or other components involved in internalization and vesicle transport. A survey of the literature indicates that, in some cases, even RTKs synthesized de novo can initiate signaling at the endoplasmic reticulum/Golgi before reaching the plasma membrane. The spectrum of molecules targeted by the signal is likely to be different between cell surface- and endoplasmic reticulum/Golgi-localized RTKs.</p>","PeriodicalId":72547,"journal":{"name":"Cellular logistics","volume":"4 ","pages":"e28461"},"PeriodicalIF":0.0,"publicationDate":"2014-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4161/cl.28461","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32659379","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-01-09eCollection Date: 2013-01-01DOI: 10.4161/cl.27609
Paul A Randazzo, Xiaoying Jian, Pei-Wen Chen, Peng Zhai, Olivier Soubias, John K Northup
The proteins that possess guanine nucleotide exchange factor (GEF) activity, which include about ~800 G protein coupled receptors (GPCRs),1 15 Arf GEFs,2 81 Rho GEFs,3 8 Ras GEFs,4 and others for other families of GTPases,5 catalyze the exchange of GTP for GDP on all regulatory guanine nucleotide binding proteins. Despite their importance as catalysts, relatively few exchange factors (we are aware of only eight for ras superfamily members) have been rigorously characterized kinetically.5-13 In some cases, kinetic analysis has been simplistic leading to erroneous conclusions about mechanism (as discussed in a recent review14). In this paper, we compare two approaches for determining the kinetic properties of exchange factors: (i) examining individual equilibria, and; (ii) analyzing the exchange factors as enzymes. Each approach, when thoughtfully used,14,15 provides important mechanistic information about the exchange factors. The analysis as enzymes is described in further detail. With the focus on the production of the biologically relevant guanine nucleotide binding protein complexed with GTP (G•GTP), we believe it is conceptually simpler to connect the kinetic properties to cellular effects. Further, the experiments are often more tractable than those used to analyze the equilibrium system and, therefore, more widely accessible to scientists interested in the function of exchange factors.
{"title":"Quantitative Analysis of Guanine Nucleotide Exchange Factors (GEFs) as Enzymes.","authors":"Paul A Randazzo, Xiaoying Jian, Pei-Wen Chen, Peng Zhai, Olivier Soubias, John K Northup","doi":"10.4161/cl.27609","DOIUrl":"https://doi.org/10.4161/cl.27609","url":null,"abstract":"<p><p>The proteins that possess guanine nucleotide exchange factor (GEF) activity, which include about ~800 G protein coupled receptors (GPCRs),<sup>1</sup> 15 Arf GEFs,<sup>2</sup> 81 Rho GEFs,<sup>3</sup> 8 Ras GEFs,<sup>4</sup> and others for other families of GTPases,<sup>5</sup> catalyze the exchange of GTP for GDP on all regulatory guanine nucleotide binding proteins. Despite their importance as catalysts, relatively few exchange factors (we are aware of only eight for ras superfamily members) have been rigorously characterized kinetically.<sup>5</sup><sup>-</sup><sup>13</sup> In some cases, kinetic analysis has been simplistic leading to erroneous conclusions about mechanism (as discussed in a recent review<sup>14</sup>). In this paper, we compare two approaches for determining the kinetic properties of exchange factors: (i) examining individual equilibria, and; (ii) analyzing the exchange factors as enzymes. Each approach, when thoughtfully used,<sup>14</sup><sup>,</sup><sup>15</sup> provides important mechanistic information about the exchange factors. The analysis as enzymes is described in further detail. With the focus on the production of the biologically relevant guanine nucleotide binding protein complexed with GTP (G•GTP), we believe it is conceptually simpler to connect the kinetic properties to cellular effects. Further, the experiments are often more tractable than those used to analyze the equilibrium system and, therefore, more widely accessible to scientists interested in the function of exchange factors.</p>","PeriodicalId":72547,"journal":{"name":"Cellular logistics","volume":"3 ","pages":"e27609"},"PeriodicalIF":0.0,"publicationDate":"2014-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4161/cl.27609","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32761038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-01-09eCollection Date: 2013-01-01DOI: 10.4161/cl.27687
Yusong Guo, Adam D Linstedt
Membrane recruitment of the COPI vesicle coat is fundamental to its function and contributes to compartment identity in the early secretory pathway. COPI recruitment is triggered by guanine nucleotide exchange activating the Arf1 GTPase, but the key exchange factor, GBF1, is a peripheral membrane component whose membrane association is dependent on another GTPase, Rab1. Inactive Rab GTPases are in a soluble complex with guanine nucleotide dissociation inhibitor (GDI) and activation of Rab GTPases by exchange factors can be enhanced by GDI dissociation factors (GDFs). In the present study, we investigated the vesicle docking protein p115 and it's binding to the Rab1 isoform Rab1b. Inhibition of p115 expression induced dissociation of Rab1b from Golgi membranes. Rab1b bound the cc2 domain of p115 and p115 lacking this domain failed to recruit Rab1b. Further, p115 inhibition blocked association of the COPI coat with Golgi membranes and this was suppressed by constitutive activation of Rab1b. These findings show p115 enhancement of Rab1b activation leading to COPI recruitment suggesting a connection between the vesicle docking machinery and the vesicle coat complex during the establishment of post-ER compartment identity.
{"title":"Binding of the vesicle docking protein p115 to the GTPase Rab1b regulates membrane recruitment of the COPI vesicle coat.","authors":"Yusong Guo, Adam D Linstedt","doi":"10.4161/cl.27687","DOIUrl":"https://doi.org/10.4161/cl.27687","url":null,"abstract":"<p><p>Membrane recruitment of the COPI vesicle coat is fundamental to its function and contributes to compartment identity in the early secretory pathway. COPI recruitment is triggered by guanine nucleotide exchange activating the Arf1 GTPase, but the key exchange factor, GBF1, is a peripheral membrane component whose membrane association is dependent on another GTPase, Rab1. Inactive Rab GTPases are in a soluble complex with guanine nucleotide dissociation inhibitor (GDI) and activation of Rab GTPases by exchange factors can be enhanced by GDI dissociation factors (GDFs). In the present study, we investigated the vesicle docking protein p115 and it's binding to the Rab1 isoform Rab1b. Inhibition of p115 expression induced dissociation of Rab1b from Golgi membranes. Rab1b bound the cc2 domain of p115 and p115 lacking this domain failed to recruit Rab1b. Further, p115 inhibition blocked association of the COPI coat with Golgi membranes and this was suppressed by constitutive activation of Rab1b. These findings show p115 enhancement of Rab1b activation leading to COPI recruitment suggesting a connection between the vesicle docking machinery and the vesicle coat complex during the establishment of post-ER compartment identity.</p>","PeriodicalId":72547,"journal":{"name":"Cellular logistics","volume":"3 ","pages":"e27687"},"PeriodicalIF":0.0,"publicationDate":"2014-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4161/cl.27687","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32761039","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-01-01Epub Date: 2014-02-13DOI: 10.4161/cl.27888
Rose Willett, Irina Pokrovskaya, Tetyana Kudlyk, Vladimir Lupashin
The conserved oligomeric Golgi complex is a peripheral membrane protein complex that orchestrates the tethering and fusion of intra-Golgi transport carriers with Golgi membranes. In this study we have investigated the membrane attachment of the COG complex and it's on/off dynamic on Golgi membranes. Several complimentary approaches including knock-sideways depletion, FRAP, and FLIP revealed that assembled COG complex is not diffusing from Golgi periphery in live HeLa cells. Moreover, COG subunits remained membrane-associated even in COG4 and COG7 depleted cells when Golgi architecture was severely affected. Overexpression of myc-tagged COG sub-complexes revealed that different membrane-associated COG partners including β-COP, p115 and SNARE STX5 preferentially bind to different COG assemblies, indicating that COG subunits interact with Golgi membranes in a multipronged fashion.
{"title":"Multipronged interaction of the COG complex with intracellular membranes.","authors":"Rose Willett, Irina Pokrovskaya, Tetyana Kudlyk, Vladimir Lupashin","doi":"10.4161/cl.27888","DOIUrl":"https://doi.org/10.4161/cl.27888","url":null,"abstract":"<p><p>The conserved oligomeric Golgi complex is a peripheral membrane protein complex that orchestrates the tethering and fusion of intra-Golgi transport carriers with Golgi membranes. In this study we have investigated the membrane attachment of the COG complex and it's on/off dynamic on Golgi membranes. Several complimentary approaches including knock-sideways depletion, FRAP, and FLIP revealed that assembled COG complex is not diffusing from Golgi periphery in live HeLa cells. Moreover, COG subunits remained membrane-associated even in COG4 and COG7 depleted cells when Golgi architecture was severely affected. Overexpression of myc-tagged COG sub-complexes revealed that different membrane-associated COG partners including β-COP, p115 and SNARE STX5 preferentially bind to different COG assemblies, indicating that COG subunits interact with Golgi membranes in a multipronged fashion.</p>","PeriodicalId":72547,"journal":{"name":"Cellular logistics","volume":"4 1","pages":"e27888"},"PeriodicalIF":0.0,"publicationDate":"2014-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4161/cl.27888","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32193350","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-01-01Epub Date: 2014-02-28DOI: 10.4161/cl.28217
Giovanni Stefano, Federica Brandizzi
The architectural integrity of the endoplasmic reticulum (ER) network depends on the function of membrane-associated dynamin-like GTPases that include metazoan atlastins, plant RHD3 and yeast Sey1p. The evidence that these proteins are sufficient to drive membrane fusion of reconstituted proteoliposomes, and that loss-of-function mutations lead to conspicuous ER shape defects indicates that atlastins, RHD3 and Sey1p promote ER membrane fusion. However, complementation experiments in reciprocal loss-of-function backgrounds have also suggested that RHD3 and Sey1p may be not functionally equivalent, supporting that ER fusion mechanisms may be not entirely conserved in eukaryotes. In this Letter, we provide a brief overview of the field as well as evidence that may explain the functional differences of the plant and yeast ER-shaping dynamin-like GTPases.
{"title":"Unique and conserved features of the plant ER-shaping GTPase RHD3.","authors":"Giovanni Stefano, Federica Brandizzi","doi":"10.4161/cl.28217","DOIUrl":"https://doi.org/10.4161/cl.28217","url":null,"abstract":"<p><p>The architectural integrity of the endoplasmic reticulum (ER) network depends on the function of membrane-associated dynamin-like GTPases that include metazoan atlastins, plant RHD3 and yeast Sey1p. The evidence that these proteins are sufficient to drive membrane fusion of reconstituted proteoliposomes, and that loss-of-function mutations lead to conspicuous ER shape defects indicates that atlastins, RHD3 and Sey1p promote ER membrane fusion. However, complementation experiments in reciprocal loss-of-function backgrounds have also suggested that RHD3 and Sey1p may be not functionally equivalent, supporting that ER fusion mechanisms may be not entirely conserved in eukaryotes. In this Letter, we provide a brief overview of the field as well as evidence that may explain the functional differences of the plant and yeast ER-shaping dynamin-like GTPases.</p>","PeriodicalId":72547,"journal":{"name":"Cellular logistics","volume":"4 1","pages":"e28217"},"PeriodicalIF":0.0,"publicationDate":"2014-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4161/cl.28217","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32327220","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-01-01Epub Date: 2014-01-09DOI: 10.4161/cl.27732
Paulina Wyrozumska, Jason W Ashley, Sasanka Ramanadham, Qinglan Liu, W Timothy Garvey, Elizabeth Sztul
Brefeldin A (BFA) is a fungal metabolite best known for its ability to inhibit activation of ADP-ribosylation factor (Arf) and thereby inhibit secretory traffic. BFA also appears to regulate the trafficking of the GLUT4 glucose transporter by inducing its relocation from intracellular stores to the cell surface. Such redistribution of GLUT4 is normally regulated by insulin-mediated signaling. Hence, we tested whether BFA may intersect with the insulin pathway. We report that BFA causes the activation of the insulin receptor (IR), IRS-1, Akt-2, and AS160 components of the insulin pathway. The response is mediated through phosphoinositol-3-kinase (PI3K) and Akt kinase since the PI3K inhibitor wortmannin and the Akt inhibitors MK2206 and perifosine inhibit the BFA effect. BFA-mediated activation of the insulin pathway results in Akt-mediated phosphorylation of the insulin-responsive transcription factor FoxO1. This leads to nuclear exclusion of FoxO1 and a decrease in transcription of the insulin-responsive gene SIRT-1. Our findings suggest novel effects for BFA in signaling and transcription, and imply that BFA has multiple intracellular targets and can be used to regulate diverse cellular responses that include vesicular trafficking, signaling and transcription.
Brefeldin A (BFA)是一种真菌代谢物,以其抑制adp核糖基化因子(Arf)的激活从而抑制分泌流量的能力而闻名。BFA似乎还通过诱导GLUT4葡萄糖转运体从细胞内储存转移到细胞表面来调节其运输。这种GLUT4的再分配通常由胰岛素介导的信号传导调节。因此,我们测试了BFA是否可能与胰岛素途径相交。我们报道BFA导致胰岛素通路中胰岛素受体(IR)、IRS-1、Akt-2和AS160组分的激活。这种反应是通过磷酸肌醇-3激酶(PI3K)和Akt激酶介导的,因为PI3K抑制剂wortmannin和Akt抑制剂MK2206和perifosine抑制BFA的作用。bfa介导的胰岛素通路激活导致akt介导的胰岛素应答转录因子fox01的磷酸化。这导致FoxO1的核排斥和胰岛素应答基因SIRT-1的转录减少。我们的研究结果表明,BFA在信号传导和转录方面具有新的作用,并暗示BFA具有多种细胞内靶点,可用于调节多种细胞反应,包括囊泡运输、信号传导和转录。
{"title":"Novel effects of Brefeldin A (BFA) in signaling through the insulin receptor (IR) pathway and regulating FoxO1-mediated transcription.","authors":"Paulina Wyrozumska, Jason W Ashley, Sasanka Ramanadham, Qinglan Liu, W Timothy Garvey, Elizabeth Sztul","doi":"10.4161/cl.27732","DOIUrl":"https://doi.org/10.4161/cl.27732","url":null,"abstract":"<p><p>Brefeldin A (BFA) is a fungal metabolite best known for its ability to inhibit activation of ADP-ribosylation factor (Arf) and thereby inhibit secretory traffic. BFA also appears to regulate the trafficking of the GLUT4 glucose transporter by inducing its relocation from intracellular stores to the cell surface. Such redistribution of GLUT4 is normally regulated by insulin-mediated signaling. Hence, we tested whether BFA may intersect with the insulin pathway. We report that BFA causes the activation of the insulin receptor (IR), IRS-1, Akt-2, and AS160 components of the insulin pathway. The response is mediated through phosphoinositol-3-kinase (PI3K) and Akt kinase since the PI3K inhibitor wortmannin and the Akt inhibitors MK2206 and perifosine inhibit the BFA effect. BFA-mediated activation of the insulin pathway results in Akt-mediated phosphorylation of the insulin-responsive transcription factor FoxO1. This leads to nuclear exclusion of FoxO1 and a decrease in transcription of the insulin-responsive gene SIRT-1. Our findings suggest novel effects for BFA in signaling and transcription, and imply that BFA has multiple intracellular targets and can be used to regulate diverse cellular responses that include vesicular trafficking, signaling and transcription.</p>","PeriodicalId":72547,"journal":{"name":"Cellular logistics","volume":"4 1","pages":"e27732"},"PeriodicalIF":0.0,"publicationDate":"2014-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4161/cl.27732","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32355652","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}