scholarly journals Aggregation state determines the localization and function of M1– and M23–aquaporin-4 in astrocytes

2014 ◽  
Vol 204 (4) ◽  
pp. 559-573 ◽  
Author(s):  
Alex J. Smith ◽  
Byung-Ju Jin ◽  
Julien Ratelade ◽  
Alan S. Verkman
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Water Channel ◽  
Aquaporin 4 ◽  
Cellular Functions ◽  
Aggregation State ◽  
Functional Roles ◽  
State Dependent ◽  
And Function

The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23–AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1–AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23–AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1– and M23–AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1–AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23–AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state–dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1– and M23–AQP4.

scholarly journals Impact of Membrane Lipids on UapA and AzgA Transporter Subcellular Localization and Activity in Aspergillus nidulans

2021 ◽  
Vol 7 (7) ◽  
pp. 514
Author(s):  
Mariangela Dionysopoulou ◽  
George Diallinas
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Aspergillus Nidulans ◽  
Membrane Lipids ◽  
Membrane Lipid ◽  
Lipid Biosynthesis ◽  
Transport Kinetics ◽  
Negative Effect ◽  
And Function

Recent biochemical and biophysical evidence have established that membrane lipids, namely phospholipids, sphingolipids and sterols, are critical for the function of eukaryotic plasma membrane transporters. Here, we study the effect of selected membrane lipid biosynthesis mutations and of the ergosterol-related antifungal itraconazole on the subcellular localization, stability and transport kinetics of two well-studied purine transporters, UapA and AzgA, in Aspergillus nidulans. We show that genetic reduction in biosynthesis of ergosterol, sphingolipids or phosphoinositides arrest A. nidulans growth after germling formation, but solely blocks in early steps of ergosterol (Erg11) or sphingolipid (BasA) synthesis have a negative effect on plasma membrane (PM) localization and stability of transporters before growth arrest. Surprisingly, the fraction of UapA or AzgA that reaches the PM in lipid biosynthesis mutants is shown to conserve normal apparent transport kinetics. We further show that turnover of UapA, which is the transporter mostly sensitive to membrane lipid content modification, occurs during its trafficking and by enhanced endocytosis, and is partly dependent on autophagy and Hect-type HulARsp5 ubiquitination. Our results point out that the role of specific membrane lipids on transporter biogenesis and function in vivo is complex, combinatorial and transporter-dependent.

scholarly journals Stoichiometry and Turnover of the Bacterial Flagellar Switch Protein FliN

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Nicolas J. Delalez ◽  
Richard M. Berry ◽  
Judith P. Armitage
Keyword(s):  
Copy Number ◽  
Motor Proteins ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Content Type ◽  
Rotation Direction ◽  
Cell Measurement ◽  
Biological Complexes ◽  
And Function

ABSTRACTSome proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, inEscherichia coli. Our results confirm that,in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments.IMPORTANCEThe flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single-cell measurement, that both the copy number and the fraction of exchanging molecules vary with the rotational bias of the rotor. When the motor is locked in counterclockwise rotation, the copy number is similar to that determined by averaged, fixed methodologies, but when locked in a clockwise direction, the number is much lower, suggesting that that the switch complex ring is incomplete. Our results suggest that motor remodeling is an important component in tuning responses and adaptation at the motor.

scholarly journals The STAGA Subunit ADA2b Is an Important Regulator of Human GCN5 Catalysis

2008 ◽  
Vol 29 (1) ◽  
pp. 266-280 ◽  
Author(s):  
Armin M. Gamper ◽  
Jaehoon Kim ◽  
Robert G. Roeder
Keyword(s):  
Saga Complex ◽  
Histone Tails ◽  
Functional Roles ◽  
Enzymatic Function ◽  
Core Histones ◽  
Human Proteins ◽  
Important Regulator ◽  
And Function

ABSTRACT Human STAGA is a multisubunit transcriptional coactivator containing the histone acetyltransferase GCN5L. Previous studies of the related yeast SAGA complex have shown that the yeast Gcn5, Ada2, and Ada3 components form a heterotrimer that is important for the enzymatic function of SAGA. Here, we report that ADA2a and ADA2b, two human homologues of yeast Ada2, each have the ability to form a heterotrimer with ADA3 and GCN5L but that only the ADA2b homologue is found in STAGA. By comparing the patterns of acetylation of several substrates, we found context-dependent requirements for ADA2b and ADA3 for the efficient acetylation of histone tails by GCN5. With human proteins, unlike yeast proteins, the acetylation of free core histones by GCN5 is unaffected by ADA2b or ADA3. In contrast, the acetylation of mononucleosomal substrates by GCN5 is enhanced by ADA2b, with no significant additional effect of ADA3, and the efficient acetylation of nucleosomal arrays (chromatin) by GCN5 requires both ADA2b and ADA3. Thus, ADA2b and ADA3 appear to act at two different levels of histone organization within chromatin to facilitate GCN5 function. Interestingly, although ADA2a forms a complex(es) with GCN5 and ADA3 both in vitro and in vivo, ADA2a-containing complexes are unable to acetylate nucleosomal H3. We have also shown the preferential recruitment of ADA2b, relative to ADA2a, to p53-dependent genes. This finding indicates that the previously demonstrated presence and function of GCN5 on these promoters reflect the action of STAGA and that the ADA2a and ADA2b paralogues have nonredundant functional roles.

scholarly journals Analysis of the roles of phosphatidylinositol-4,5-bisphosphate and individual subunits in assembly, localization, and function of Saccharomyces cerevisiae target of rapamycin complex 2

2019 ◽  
Vol 30 (12) ◽  
pp. 1555-1574 ◽  
Author(s):  
Maria Nieves Martinez Marshall ◽  
Anita Emmerstorfer-Augustin ◽  
Kristin L. Leskoske ◽  
Lydia H. Zhang ◽  
Biyun Li ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Lipid Metabolism ◽  
Eukaryotic Cell ◽  
Target Of Rapamycin ◽  
Multiprotein Complex ◽  
Ph Domain ◽  
Evolutionarily Conserved ◽  
And Function

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2-­associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5- bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or cause disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.

Cellular structure and function

2021 ◽  
pp. 1-104
Keyword(s):  
Plasma Membrane ◽  
Nucleic Acids ◽  
Cellular Structure ◽  
Structure And Function ◽  
The Body ◽  
Signalling Pathways ◽  
Cellular Functions ◽  
Pharmacological Agents ◽  
Intracellular Organelles ◽  
And Function

‘Cellular structure and function’ covers the roles, structures, and functions of the main four types of macromolecules of the human body, namely proteins, lipids, carbohydrates, and nucleic acids. For these macromolecules, the roles and types of each class are discussed (for proteins this includes their roles as structural proteins and enzymes and their kinetics; for lipids, the roles and types of lipid found in the body are considered; for carbohydrates, their roles including structural and metabolic are discussed; and the structure of nucleic acids is described). Then follows a description of the organization of the cell, including the plasma membrane and its components, and the intracellular organelles. Cell growth, division, and apoptosis are covered, as are the formation of gametes, and finally the principles of how cellular functions can be modulated by pharmacological agents through receptors and signalling pathways are discussed.

scholarly journals Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival

2012 ◽  
Vol 199 (7) ◽  
pp. 1145-1158 ◽  
Author(s):  
Hyung Joon Kim ◽  
Vikram Prasad ◽  
Seok-Won Hyung ◽  
Zang Hee Lee ◽  
Sang-Won Lee ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Bone Mass ◽  
Osteoclast Differentiation ◽  
Premenopausal Women ◽  
Fine Tuning ◽  
Bone Homeostasis ◽  
Regulatory Loop ◽  
And Function

The precise regulation of Ca2+ dynamics is crucial for proper differentiation and function of osteoclasts. Here we show the involvement of plasma membrane Ca2+ ATPase (PMCA) isoforms 1 and 4 in osteoclastogenesis. In immature/undifferentiated cells, PMCAs inhibited receptor activator of NF-κB ligand–induced Ca2+ oscillations and osteoclast differentiation in vitro. Interestingly, nuclear factor of activated T cell c1 (NFATc1) directly stimulated PMCA transcription, whereas the PMCA-mediated Ca2+ efflux prevented NFATc1 activation, forming a negative regulatory loop. PMCA4 also had an anti-osteoclastogenic effect by reducing NO, which facilitates preosteoclast fusion. In addition to their role in immature cells, increased expression of PMCAs in mature osteoclasts prevented osteoclast apoptosis both in vitro and in vivo. Mice heterozygous for PMCA1 or null for PMCA4 showed an osteopenic phenotype with more osteoclasts on bone surface. Furthermore, PMCA4 expression levels correlated with peak bone mass in premenopausal women. Thus, our results suggest that PMCAs play important roles for the regulation of bone homeostasis in both mice and humans by modulating Ca2+ signaling in osteoclasts.

scholarly journals Genetic interaction mapping informs integrative structure determination of protein complexes

Science ◽  
2020 ◽  
Vol 370 (6522) ◽  
pp. eaaz4910 ◽  
Author(s):  
Hannes Braberg ◽  
Ignacia Echeverria ◽  
Stefan Bohn ◽  
Peter Cimermancic ◽  
Anthony Shiver ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Structure Determination ◽  
Genetic Interaction ◽  
Protein Complexes ◽  
Point Mutations ◽  
Genetic Interactions ◽  
Cellular Functions ◽  
Cross Links

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.

Regulation of microRNA biogenesis and function

2012 ◽  
Vol 107 (04) ◽  
pp. 605-610 ◽  
Author(s):  
Thomas Treiber ◽  
Nora Treiber ◽  
Gunter Meister
Keyword(s):  
Mammalian Cells ◽  
Untranslated Region ◽  
Protein Complexes ◽  
Mature Mirnas ◽  
Review Article ◽  
Cellular Functions ◽  
Target Sites ◽  
Cellular Pathways ◽  
And Function ◽  
Site Binding

SummaryMicroRNAs (miRNAs) are considered as key regulators of literally all cellular pathways. Therefore, miRNA biosynthesis and their individual cellular functions must be tightly regulated as well. MiRNAs are transcribed as primary transcripts, which are processed to mature miRNAs in two consecutive maturation steps. Finally, the mature miRNA is incorporated into a miRNA-protein complex, where it directly interacts with a member of the Argonaute (Ago) protein family. The miRNA guides such protein complexes to partial complementary target sites, which are typically located in the 3’ untranslated region (UTR) of mRNAs leading to inhibition of gene expression. MiRNA activity and abundance is regulated on various levels ranging from transcription and processing to target site binding and miRNA stability. Recent advances in our understanding of how miRNA activity is regulated in mammalian cells are summarised and discussed in this review article.

scholarly journals A synthesis of over 9,000 mass spectrometry experiments reveals the core set of human protein complexes

2016 ◽  
Author(s):  
Kevin Drew ◽  
Chanjae Lee ◽  
Ryan L. Huizar ◽  
Fan Tu ◽  
Blake Borgeson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Genetic Diseases ◽  
Intraflagellar Transport ◽  
Human Protein ◽  
Disease Genes ◽  
Cellular Functions ◽  
The Core

AbstractMacromolecular protein complexes carry out many of the essential functions of cells, and many genetic diseases arise from disrupting the functions of such complexes. Currently there is great interest in defining the complete set of human protein complexes, but recent published maps lack comprehensive coverage. Here, through the synthesis of over 9,000 published mass spectrometry experiments, we present hu.MAP, the most comprehensive and accurate human protein complex map to date, containing >4,600 total complexes, >7,700 proteins and >56,000 unique interactions, including thousands of confident protein interactions not identified by the original publications. hu.MAP accurately recapitulates known complexes withheld from the learning procedure, which was optimized with the aid of a new quantitative metric (k-cliques) for comparing sets of sets. The vast majority of complexes in our map are significantly enriched with literature annotations and the map overall shows improved coverage of many disease-associated proteins, as we describe in detail for ciliopathies. Using hu.MAP, we predicted and experimentally validated candidate ciliopathy disease genes in vivo in a model vertebrate, discovering CCDC138, WDR90, and KIAA1328 to be new cilia basal body/centriolar satellite proteins, and identifying ANKRD55 as a novel member of the intraflagellar transport machinery. By offering significant improvements to the accuracy and coverage of human protein complexes, hu.MAP (http://proteincomplexes.org) serves as a valuable resource for better understanding the core cellular functions of human proteins and helping to determine mechanistic foundations of human disease.

scholarly journals Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

2020 ◽  
Author(s):  
M. Alessandra Vigano ◽  
Clara-Maria Ell ◽  
Manuela MM Kustermann ◽  
Gustavo Aguilar ◽  
Shinya Matsuda ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Single Copy ◽  
Specific Protein ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Peptide Tags ◽  
Genetic Technologies ◽  
Protein Binders ◽  
And Function

AbstractCellular development and specialized cellular functions are regulated processes which rely on highly dynamic molecular interactions among proteins, distributed in all cell compartments. Analysis of these interactions and their mechanisms of action has been one of the main topics in cellular and developmental research over the last fifty years. Studying and understanding the functions of proteins of interest (POIs) has been mostly achieved by their alteration at the genetic level and the analysis of the phenotypic changes generated by these alterations. Although genetic and reverse genetic technologies contributed to the vast majority of information and knowledge we have gathered so far, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would require more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, together with several improvements in synthetic biology techniques, have contributed to the creation of a new toolbox for direct protein manipulations. We selected a number of short tag epitopes for which protein binders from different scaffolds have been developed and tested whether these tags can be bound by the corresponding protein binders in living cells when they are inserted in a single copy in a POI. We indeed find that in all cases, a single copy of a short tag allows protein binding and manipulation. Using Drosophila, we also find that single short tags can be recognized and allow degradation and relocalization of POIs in vivo.

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