scholarly journals Membrane Tubule-mediated Reassembly and Maintenance of the Golgi Complex Is Disrupted by Phospholipase A2Antagonists

1999 ◽  
Vol 10 (6) ◽  
pp. 1763-1782 ◽  
Author(s):  
Paul de Figueiredo ◽  
Renée S. Polizotto ◽  
Daniel Drecktrah ◽  
William J. Brown
Keyword(s):  
Steady State ◽  
Golgi Complex ◽  
Membrane Trafficking ◽  
Biological Significance ◽  
Vitro Assay ◽  
In Vitro Reconstitution ◽  
Dynamic Maintenance ◽  
Membrane Tubules ◽  
Insight Into

Although membrane tubules can be found extending from, and associated with, the Golgi complex of eukaryotic cells, their physiological function has remained unclear. To gain insight into the biological significance of membrane tubules, we have developed methods for selectively preventing their formation. We show here that a broad range of phospholipase A2(PLA2) antagonists not only arrest membrane tubule–mediated events that occur late in the assembly of the Golgi complex but also perturb its normal steady-state tubulovesicular architecture by inducing a reversible fragmentation into separate “mini-stacks.” In addition, we show that these same compounds prevent the formation of membrane tubules from Golgi stacks in an in vitro reconstitution system. This in vitro assay was further used to demonstrate that the relevant PLA2activity originates from the cytoplasm. Taken together, these results demonstrate that Golgi membrane tubules, sensitive to potent and selective PLA2antagonists, mediate both late events in the reassembly of the Golgi complex and the dynamic maintenance of its steady-state architecture. In addition, they implicate a role for cytoplasmic PLA2enzymes in mediating these membrane trafficking events.

scholarly journals Microtubule minus-end stability is dictated by the tubulin off-rate

2019 ◽  
Vol 218 (9) ◽  
pp. 2841-2853 ◽  
Author(s):  
Claire Strothman ◽  
Veronica Farmer ◽  
Göker Arpağ ◽  
Nicole Rodgers ◽  
Marija Podolski ◽  
...  
Keyword(s):  
Dynamic Instability ◽  
In Vitro Reconstitution ◽  
Primary Determinant ◽  
Mean Size ◽  
The Mean ◽  
The Difference ◽  
Insight Into ◽  
Dynamic Organization ◽  
In Cells

Dynamic organization of microtubule minus ends is vital for the formation and maintenance of acentrosomal microtubule arrays. In vitro, both microtubule ends switch between phases of assembly and disassembly, a behavior called dynamic instability. Although minus ends grow slower, their lifetimes are similar to those of plus ends. The mechanisms underlying these distinct dynamics remain unknown. Here, we use an in vitro reconstitution approach to investigate minus-end dynamics. We find that minus-end lifetimes are not defined by the mean size of the protective GTP-tubulin cap. Rather, we conclude that the distinct tubulin off-rate is the primary determinant of the difference between plus- and minus-end dynamics. Further, our results show that the minus-end–directed kinesin-14 HSET/KIFC1 suppresses tubulin off-rate to specifically suppress minus-end catastrophe. HSET maintains its protective minus-end activity even when challenged by a known microtubule depolymerase, kinesin-13 MCAK. Our results provide novel insight into the mechanisms of minus-end dynamics, essential for our understanding of microtubule minus-end regulation in cells.

scholarly journals Septin 2/6/7 complexes tune microtubule plus-end growth and EB1 binding in a concentration- and filament-dependent manner

2019 ◽  
Vol 30 (23) ◽  
pp. 2913-2928 ◽  
Author(s):  
Konstantinos Nakos ◽  
Megan R. Radler ◽  
Elias T. Spiliotis
Keyword(s):  
Membrane Trafficking ◽  
Binding Proteins ◽  
Live Cells ◽  
Dependent Manner ◽  
Guanosine Diphosphate ◽  
In Vitro Reconstitution ◽  
In Trans ◽  
In Cis ◽  
Precise Role

Septins (SEPTs) are filamentous guanosine-5′-triphosphate (GTP)-binding proteins, which affect microtubule (MT)-dependent functions including membrane trafficking and cell division, but their precise role in MT dynamics is poorly understood. Here, in vitro reconstitution of MT dynamics with SEPT2/6/7, the minimal subunits of septin heteromers, shows that SEPT2/6/7 has a biphasic concentration-dependent effect on MT growth. Lower concentrations of SEPT2/6/7 enhance MT plus-end growth and elongation, while higher and intermediate concentrations inhibit and pause plus-end growth, respectively. We show that SEPT2/6/7 has a modest preference for GTP- over guanosine diphosphate (GDP)-bound MT lattice and competes with end-binding protein 1 (EB1) for binding to guanosine 5′- O-[γ-thio]triphosphate (GTPγS)-stabilized MTs, which mimic the EB1-preferred GDP-Pi state of polymerized tubulin. Strikingly, SEPT2/6/7 triggers EB1 dissociation from plus-end tips in cis by binding to the MT lattice and in trans when MT plus ends collide with SEPT2/6/7 filaments. At these intersections, SEPT2/6/7 filaments were more potent barriers than actin filaments in pausing MT growth and dissociating EB1 in vitro and in live cells. These data demonstrate that SEPT2/6/7 complexes and filaments can directly impact MT plus-end growth and the tracking of plus end–binding proteins and thereby may facilitate the capture of MT plus ends at intracellular sites of septin enrichment. [Media: see text]

BIOLOGICAL SIGNIFICANCE OF ACTIVE AND INACTIVE RENIN IN MAN

1980 ◽  
Vol 85 (1) ◽  
pp. 137-143 ◽  
Author(s):  
P. LIJNEN ◽  
A. AMERY ◽  
R. FAGARD ◽  
L. VERSCHUEREN
Keyword(s):  
Plasma Concentrations ◽  
Biological Significance ◽  
Plasma Renin ◽  
Angiotensin I ◽  
Vitro Assay ◽  
Arterial Blood ◽  
Inactive Renin ◽  
Change In Mean

SUMMARY The biological significance of active and inactive renin was investigated by comparison of an in-vitro assay of active, total and inactive plasma renin concentration (PRC), plasma renin activity (PRA) and plasma concentrations of angiotensin I and II with an in-vivo change in mean arterial blood pressure (MAP) produced by antagonism of angiotensin with treatment with saralasin and by blockade of angiotensin-converting enzyme by treatment with captopril. A significant relationship between the changes in MAP during treatment with saralasin and captopril with the pretreatment levels of PRA, active and total PRC and angiotensin II were found; while the pre-existing level of inactive renin was not a predictor for the hypotensive effect of saralasin and captopril. During treatment with saralasin and captopril significant increases in PRA, plasma angiotensin I concentration and total and active PRC were found and no change in inactive PRC was observed.

scholarly journals Autoregulation Is Essential for Precise Temporal and Steady-State Regulation by the Bordetella BvgAS Phosphorelay

2006 ◽  
Vol 189 (5) ◽  
pp. 1974-1982 ◽  
Author(s):  
Corinne L. Williams ◽  
Peggy A. Cotter
Keyword(s):  
Gene Expression ◽  
Steady State ◽  
Response Regulator ◽  
Expression Patterns ◽  
State Regulation ◽  
Temporal Expression ◽  
Multiple Gene ◽  
Insight Into

ABSTRACT The Bordetella BvgAS virulence control system is prototypical of phosphorelays that use a polydomain sensor and a response regulator to control gene expression in response to environmental cues. BvgAS controls the expression of at least three distinct phenotypic phases (Bvg−, Bvgi, and Bvg+) by differentially regulating the expression of at least four classes of genes. Among the loci regulated by BvgAS is bvgAS itself. We investigated the role of autoregulation in the ability of BvgAS to control multiple gene expression patterns in a temporal and steady-state manner by constructing Bordetella bronchiseptica strains in which the bvgAS promoter was replaced with constitutively active promoters. Our results show that positive autoregulation of bvgAS transcription is required for the temporal expression of multiple phenotypic phases that occurs in response to a shift from Bvg−-phase conditions to Bvg+-phase conditions. Autoregulation was also shown to contribute to steady-state regulation; it influences the sensitivity of the system in response to subtle differences in signal intensity. In addition, considered in relation to BvgA and BvgS activities demonstrated in vitro, our results provide insight into how BvgA and BvgS function mechanistically.

scholarly journals Transmembrane helix hydrophobicity is an energetic barrier during the retrotranslocation of integral membrane ERAD substrates

2017 ◽  
Vol 28 (15) ◽  
pp. 2076-2090 ◽  
Author(s):  
Christopher J. Guerriero ◽  
Karl-Richard Reutter ◽  
Andrew A. Augustine ◽  
G. Michael Preston ◽  
Kurt F. Weiberth ◽  
...  
Keyword(s):  
Molecular Chaperones ◽  
Transmembrane Helix ◽  
Integral Membrane Proteins ◽  
Membrane Insertion ◽  
Transmembrane Helices ◽  
Vitro Assay ◽  
Dual Pass ◽  
Erad Pathway ◽  
Insight Into

Integral membrane proteins fold inefficiently and are susceptible to turnover via the endoplasmic reticulum–associated degradation (ERAD) pathway. During ERAD, misfolded proteins are recognized by molecular chaperones, polyubiquitinated, and retrotranslocated to the cytoplasm for proteasomal degradation. Although many aspects of this pathway are defined, how transmembrane helices (TMHs) are removed from the membrane and into the cytoplasm before degradation is poorly understood. In this study, we asked whether the hydrophobic character of a TMH acts as an energetic barrier to retrotranslocation. To this end, we designed a dual-pass model ERAD substrate, Chimera A*, which contains the cytoplasmic misfolded domain from a characterized ERAD substrate, Sterile 6* (Ste6p*). We found that the degradation requirements for Chimera A* and Ste6p* are similar, but Chimera A* was retrotranslocated more efficiently than Ste6p* in an in vitro assay in which retrotranslocation can be quantified. We then constructed a series of Chimera A* variants containing synthetic TMHs with a range of ΔG values for membrane insertion. TMH hydrophobicity correlated inversely with retrotranslocation efficiency, and in all cases, retrotranslocation remained Cdc48p dependent. These findings provide insight into the energetic restrictions on the retrotranslocation reaction, as well as a new computational approach to predict retrotranslocation efficiency.

scholarly journals ARF1 dimerization is essential for vesicle trafficking and dependent on activation by ARF-GEF dimers in Arabidopsis

2020 ◽  
Author(s):  
Sabine Brumm ◽  
Mads Eggert Nielsen ◽  
Sandra Richter ◽  
Hauke Beckmann ◽  
York-Dieter Stierhof ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Membrane Vesicle ◽  
Biological Significance ◽  
Eukaryotic Cell ◽  
Vesicle Formation ◽  
Coat Proteins ◽  
Nucleotide Exchange ◽  
Sites Of Action

AbstractMembrane traffic maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations such as sites of action or degradation. Membrane vesicle formation requires ARF GTPase activation by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs have been shown to form dimers in vivo. This feature is conserved across the eukaryotes, however its biological significance is unknown. Here we demonstrate ARF1 dimerization in vivo and we show that ARF-GEF dimers mediate ARF1 dimer formation. Mutational disruption of ARF1 dimers interfered with ARF1-dependent trafficking but not coat protein recruitment in Arabidopsis. Mutations disrupting simultaneous binding of two ARF1•GDPs by the two SEC7 domains of GNOM ARF-GEF dimer prevented stable interaction of ARF1 with ARF-GEF and thus, efficient ARF1 activation. Our results suggest a model of activation-dependent dimerization of membrane-inserted ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

scholarly journals Bifidobacterium response to lactulose ingestion in the gut relies on a solute-binding protein-dependent ABC transporter

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Keisuke Yoshida ◽  
Rika Hirano ◽  
Yohei Sakai ◽  
Moonhak Choi ◽  
Mikiyasu Sakanaka ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Binding Protein ◽  
Bifidobacterium Longum ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Mutant Strains ◽  
Mechanistic Basis ◽  
Insight Into ◽  
Japanese Subjects

AbstractThis study aims to understand the mechanistic basis underlying the response of Bifidobacterium to lactulose ingestion in guts of healthy Japanese subjects, with specific focus on a lactulose transporter. An in vitro assay using mutant strains of Bifidobacterium longum subsp. longum 105-A shows that a solute-binding protein with locus tag number BL105A_0502 (termed LT-SBP) is primarily involved in lactulose uptake. By quantifying faecal abundance of LT-SBP orthologues, which is defined by phylogenetic analysis, we find that subjects with 107 to 109 copies of the genes per gram of faeces before lactulose ingestion show a marked increase in Bifidobacterium after ingestion, suggesting the presence of thresholds between responders and non-responders to lactulose. These results help predict the prebiotics-responder and non-responder status and provide an insight into clinical interventions that test the efficacy of prebiotics.

scholarly journals Septin 2/6/7 complexes tune microtubule plus end growth and EB1 binding in a concentration- and filament-dependent manner

2019 ◽  
Author(s):  
Konstantinos Nakos ◽  
Megan R. Radler ◽  
Elias T. Spiliotis
Keyword(s):  
Membrane Trafficking ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Live Cells ◽  
Dependent Manner ◽  
In Vitro Reconstitution ◽  
In Trans ◽  
In Cis ◽  
Precise Role

AbstractSeptins are filamentous GTP-binding proteins, which affect microtubule (MT) dependent functions including membrane trafficking and cell division, but their precise role in MT dynamics is poorly understood. Here, in vitro reconstitution of MT dynamics with SEPT2/6/7, the minimal subunits of septin heteromers, shows that SEPT2/6/7 has a biphasic concentration-dependent effect on MT growth. Lower concentrations of SEPT2/6/7 enhance MT plus end growth and elongation, while higher and intermediate concentrations inhibit and pause plus end growth, respectively. We show that SEPT2/6/7 has a 1.5-fold preference for GTP-over GDP-bound MT lattice, and competes with EB1 for binding to GTPγS-stabilized MTs, which mimic the EB1-preferred GDP-Pi state of polymerized tubulin. Strikingly, SEPT2/6/7 triggers EB1 dissociation from plus end tips in cis by binding to the MT lattice and in trans when MT plus ends collide with SEPT2/6/7 filaments. At these intersections, SEPT2/6/7 filaments were more potent barriers than actin filaments in pausing MT growth and dissociating EB1 in vitro and in live cells. These data demonstrate that SEPT2/6/7 complexes and filaments can directly impact MT plus end growth and the tracking of plus end-binding proteins, and thereby may facilitate the capture of MT plus ends at intracellular sites of septin enrichment.Highlight Summary for eTOCKnowledge of septin roles in MT dynamics is poor and confounded by knockdown studies. Here, in vitro reconstitution assays show concentration-dependent effects of SEPT2/6/7 on MT plus end growth, pausing and EB1 tracking. We found that SEPT2/6/7 filaments are potent than actin in pausing MT growth and dissociating EB1 from intersecting plus ends.

GAIP, a Gαi-3-binding protein, is associated with Golgi-derived vesicles and protein trafficking

1999 ◽  
Vol 276 (2) ◽  
pp. C497-C506 ◽  
Author(s):  
Fiona Wylie ◽  
Kirsten Heimann ◽  
Tam Luan Le ◽  
Darren Brown ◽  
Glenn Rabnott ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Membrane Trafficking ◽  
Protein Trafficking ◽  
Protein Signaling ◽  
Interacting Protein ◽  
Vitro Assay ◽  
Activated State ◽  
Show Evidence ◽  
Golgi Network

Proteins of the regulators of G protein signaling (RGS) family bind to Gα subunits to downregulate their signaling in a variety of systems. Gα-interacting protein (GAIP) is a mammalian RGS protein that shows high affinity for the activated state of Gαi-3, a protein known to regulate post-Golgi trafficking of secreted proteins in kidney epithelial cells. This study aimed to localize GAIP in epithelial cells and to investigate its potential role in the regulation of membrane trafficking. LLC-PK1 cells were stably transfected with a c- myc-tagged GAIP cDNA. In the transfected and untransfected cells, GAIP was found in the cytosol and on cell membranes. Immunogold labeling showed that membrane-bound GAIP was localized on budding vesicles around Golgi stacks. When an in vitro assay was used to generate vesicles from isolated rat liver and Madin-Darby canine kidney cell Golgi membranes, GAIP was found to be concentrated in fractions of newly budded Golgi vesicles. Finally, the constitutive trafficking and secretion of sulfated proteoglycans was measured in cell lines overexpressing GAIP. We show evidence for GAIP regulation of secretory trafficking before the level of the trans-Golgi network but not in post-Golgi secretion. The location and functional effects of GAIP overlap only partially with those of Gαi-3 and suggest multiple roles for GAIP in epithelial cells.

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