Sequence Analysis of a Subset of Plasma Membrane Raft Proteome Containing CXXC Metal Binding Motifs

2015 ◽  
Vol 5 (2) ◽  
pp. 1-15
Author(s):  
Santosh Kumar Sahu ◽  
Himadri Gourav Behuria ◽  
Sangam Gupta ◽  
Babita Sahoo
Keyword(s):  
Plasma Membrane ◽  
Metal Binding ◽  
Mammalian Cells ◽  
Membrane Raft ◽  
Binding Motifs ◽  
Cxxc Motif ◽  
Signaling Mechanisms ◽  
Associated Proteins ◽  
Heavy Metal Sensing ◽  
Metal Sensing

In an attempt to identify the metal sensing proteins localized to mammalian plasma membrane, the authors screened a list of 300 raft associated proteins that are involved in cellular signaling mechanisms by searching the presence of metal thionin (CXXC) motifs. 50 proteins were found to possess CXXC motifs that could act as potential metal sensing proteins. The authors determined membrane topologies of the above CXXC motif containing proteins using TM-pred and analyzed the positions of their transmembrane (TM) domains using Bio-edit software. Based on the topology of CXXC domains, the authors classified all the raft-associated metal sensing proteins into six categories. They are (i) Exoplasmic tails with CXXC motif, (ii) Exoplasmic loops with CXXC motif, (iii) Cytosolic tails with CXXC motif, (iv) Cytosolic loop with CXXC motif, (v) TM domains with CXXC motifs, (vi) Proteins with multiple topologies of CXXC motif. The authors' study will lead to understanding of the raft-mediated mechanism of heavy metal sensing and signaling in mammalian cells.

scholarly journals The eisosome core is composed of BAR domain proteins

2011 ◽  
Vol 22 (13) ◽  
pp. 2360-2372 ◽  
Author(s):  
Agustina Olivera-Couto ◽  
Martin Graña ◽  
Laura Harispe ◽  
Pablo S. Aguilar
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Site Directed Mutagenesis ◽  
Bar Domain ◽  
Macromolecular Assemblies ◽  
Surface Patches ◽  
Cytoplasmic Proteins ◽  
Associated Proteins ◽  
Core Components ◽  
Membrane Bending

Eisosomes define sites of plasma membrane organization. In Saccharomyces cerevisiae, eisosomes delimit furrow-like plasma membrane invaginations that concentrate sterols, transporters, and signaling molecules. Eisosomes are static macromolecular assemblies composed of cytoplasmic proteins, most of which have no known function. In this study, we used a bioinformatics approach to analyze a set of 20 eisosome proteins. We found that the core components of eisosomes, paralogue proteins Pil1 and Lsp1, are distant homologues of membrane-sculpting Bin/amphiphysin/Rvs (BAR) proteins. Consistent with this finding, purified recombinant Pil1 and Lsp1 tubulated liposomes and formed tubules when the proteins were overexpressed in mammalian cells. Structural homology modeling and site-directed mutagenesis indicate that Pil1 positively charged surface patches are needed for membrane binding and liposome tubulation. Pil1 BAR domain mutants were defective in both eisosome assembly and plasma membrane domain organization. In addition, we found that eisosome-associated proteins Slm1 and Slm2 have F-BAR domains and that these domains are needed for targeting to furrow-like plasma membrane invaginations. Our results support a model in which BAR domain protein–mediated membrane bending leads to clustering of lipids and proteins within the plasma membrane.

scholarly journals MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration

2011 ◽  
Vol 22 (8) ◽  
pp. 1252-1262 ◽  
Author(s):  
Juan F. Aranda ◽  
Natalia Reglero-Real ◽  
Leonor Kremer ◽  
Beatriz Marcos-Ramiro ◽  
Ana Ruiz-Sáenz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Migration ◽  
Mammalian Cells ◽  
Cell Spreading ◽  
Membrane Domains ◽  
Membrane Raft ◽  
General Process ◽  
Membrane Protrusions ◽  
Differentiation Marker ◽  
And Migration

Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes.

scholarly journals Functional Analysis of the N-terminal CXXC Metal-binding Motifs in the Human Menkes Copper-transporting P-type ATPase Expressed in Cultured Mammalian Cells

1999 ◽  
Vol 274 (31) ◽  
pp. 22008-22012 ◽  
Author(s):  
Ilia Voskoboinik ◽  
Daniel Strausak ◽  
Mark Greenough ◽  
Hilary Brooks ◽  
Michael Petris ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Metal Binding ◽  
Mammalian Cells ◽  
Binding Motifs ◽  
P Type

scholarly journals Lipid-modified, cysteinyl-containing peptides of diverse structures are efficiently S-acylated at the plasma membrane of mammalian cells.

1996 ◽  
Vol 134 (3) ◽  
pp. 647-660 ◽  
Author(s):  
H Schroeder ◽  
R Leventis ◽  
S Shahinian ◽  
P A Walton ◽  
J R Silvius
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Spectrometric Analysis ◽  
Acylation Reaction ◽  
Preferential Localization ◽  
Membrane Compartment ◽  
Associated Proteins ◽  
Modified Peptides ◽  
Almost All

A variety of cysteine-containing, lipid-modified peptides are found to be S-acylated by cultured mammalian cells. The acylation reaction is highly specific for cysteinyl over serinyl residues and for lipid-modified peptides over hydrophilic peptides. The S-acylation process appears by various criteria to be enzymatic and resembles the S-acylation of plasma membrane-associated proteins in various characteristics, including inhibition by tunicamycin. The substrate range of the S-acylation reaction encompasses, but is not limited to, lipopeptides incorporating the motifs myristoylGC- and -CXC(farnesyl)-OCH3, which are reversibly S-acylated in various intracellular proteins. Mass-spectrometric analysis indicates that palmitoyl residues constitute the predominant but not the only type of S-acyl group coupled to a lipopeptide carrying the myristoylGC- motif, with smaller amounts of S-stearoyl and S-oleoyl substituents also detectable. Fluorescence microscopy using NBD-labeled cysteinyl lipopeptides reveals that the products of lipopeptide S-acylation, which cannot diffuse between membranes, are in almost all cases localized preferentially to the plasma membrane. This preferential localization is found even at reduced temperatures where vesicular transport from the Golgi complex to the plasma membrane is suppressed, strongly suggesting that the plasma membrane itself is the preferred site of S-acylation of these species. Uniquely among the lipopeptides studied, species incorporating an unphysiological N-myristoylcysteinyl- motif also show substantial formation of S-acylated products in a second, intracellular compartment identified as the Golgi complex by its labeling with a fluorescent ceramide. Our results suggest that distinct S-acyltransferases exist in the Golgi complex and plasma membrane compartments and that S-acylation of motifs such as myristoylGC- occurs specifically at the plasma membrane, affording efficient targeting of cellular proteins bearing such motifs to this membrane compartment.

TRASH: a novel metal-binding domain predicted to be involved in heavy-metal sensing, trafficking and resistance

2003 ◽  
Vol 28 (4) ◽  
pp. 170-173 ◽  
Author(s):  
Thijs J.G. Ettema ◽  
Martijn A. Huynen ◽  
Willem M. de Vos ◽  
John van der Oost
Keyword(s):  
Heavy Metal ◽  
Metal Binding ◽  
Binding Domain ◽  
Metal Binding Domain ◽  
Heavy Metal Sensing ◽  
Metal Sensing

scholarly journals Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells.

1999 ◽  
Vol 274 (50) ◽  
pp. 36030
Author(s):  
Ilia Voskoboinik ◽  
Daniel Strausak ◽  
Mark Greenough ◽  
Hilary Brooks ◽  
Michael Petris ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Metal Binding ◽  
Mammalian Cells ◽  
Binding Motifs ◽  
P Type

Biotinylation and Purification of Plasma Membrane-associated Proteins from Rodent Cultured Neurons

BIO-PROTOCOL ◽  
2016 ◽  
Vol 6 (10) ◽  
Author(s):  
Margarida Caldeira ◽  
Joana Ferreira ◽  
Ana Carvalho ◽  
Carlos Duarte
Keyword(s):  
Plasma Membrane ◽  
Cultured Neurons ◽  
Associated Proteins

scholarly journals Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1834
Author(s):  
Tomoko Okada ◽  
Toshihiko Ogura
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Related Gene ◽  
Autophagosome Formation ◽  
Intact Cells ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Difference ◽  
Related Proteins ◽  
Scanning Electron

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

scholarly journals The polymerization of actin: II. how nonfilamentous actin becomes nonrandomly distributed in sperm: evidence for the association of this actin with membranes

1976 ◽  
Vol 69 (1) ◽  
pp. 51-72 ◽  
Author(s):  
LG Tilney
Keyword(s):  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Thin Section ◽  
Actin Filaments ◽  
Early Stage ◽  
Mature Sperm ◽  
Vacuole Membrane ◽  
Other Substances ◽  
Associated Proteins ◽  
Basal Half

At an early stage in spermiogenesis the acrosomal vacuole and other organelles including ribosomes are located at the basal end of the cell. From here actin must be transported to its future location at the anterior end of the cell. At no stage in the accumulation of actin in the periacrosomal region is the actin sequestered in a membrane-bounded compartment such as a vacuole or vesicle. Since filaments are not present in the periacrosomal region during the accumulation of the actin even though the fixation of these cells is sufficiently good to distinguish actin filaments in thin section, the actin must accumulate in the nonfilamentous state. The membranes in the periacrosomal region, specifically a portion of the nuclear envelope and the basal half of the acrosomal vacuole membrane, become specialized morphologically in advance of the accumulation of actin in this region. My working hypothesis is that the actin in combination with other substances binds to these specialized membranes and to itself and thus can accumulate in the periacrosmoal region by being trapped on these specialized membranes. Diffusion would then be sufficient to move these substances to this region. In support of this hypothesis are experiments in which I treated mature sperm with detergents, glycols, and hypotonic media, which solubilize or lift away the plasma membrane. The actin and its associated proteins remain attached to these specialized membranes. Thus actin can be nonrandomly distributed in cells in a nonfilamentous state presumably by its association with specialized membranes.

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