scholarly journals Functional Significance of Conserved Cysteines in the Extracellular Loops of the ATP Binding Cassette Transporter Pdr11p

2020 ◽  
Vol 7 (1) ◽  
pp. 2
Author(s):  
Lyubomir Dimitrov Stanchev ◽  
Magdalena Marek ◽  
Feng Xian ◽  
Mara Klöhn ◽  
Daniele Silvestro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Mass Spectrometry Analysis ◽  
Cysteine Residues ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutant Proteins ◽  
Spectrometry Analysis ◽  
Sterol Uptake ◽  
Extracellular Loops

The pleiotropic drug resistance (PDR) transporter Pdr11p is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae, where it facilitates the uptake of exogenous sterols. Members of the fungal PDR family contain six conserved cysteines in their extracellular loops (ECL). For the functional analysis of these cysteine residues in Pdr11p, we generated a series of single cysteine-to-serine mutants. All mutant proteins expressed well and displayed robust ATPase activity upon purification. Mass-spectrometry analysis identified two cysteine residues (C582 and C603) in ECL3 forming a disulfide bond. Further characterization by cell-based assays showed that all mutants are compromised in facilitating sterol uptake, protein stability, and trafficking to the plasma membrane. Our data highlight the fundamental importance of all six extracellular cysteine residues for the functional integrity of Pdr11p and provide new structural insights into the PDR family of transporters.

scholarly journals Sperm acrosin is responsible for the sperm binding to the egg envelope during fertilization in Japanese quail (Coturnix japonica)

Reproduction ◽  
2011 ◽  
Vol 142 (2) ◽  
pp. 267-276 ◽  
Author(s):  
Tomohiro Sasanami ◽  
Norio Yoshizaki ◽  
Hideo Dohra ◽  
Hideo Kubo
Keyword(s):  
Plasma Membrane ◽  
Japanese Quail ◽  
Sodium Dodecyl ◽  
Periodate Oxidation ◽  
Apparent Molecular Weight ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Sperm Binding ◽  
Sperm Plasma Membrane ◽  
Membrane Components

An antibody library against quail sperm plasma membrane components was established and a mAb, which strongly inhibits sperm perforations of the perivitelline membrane (PVM) was obtained from the library. The antigen molecule of the mAb showed an apparent molecular weight of 45 kDa, and was distributed both on the surface and in the acrosomal matrix of the sperm head. Periodate oxidation revealed that the epitope of the antigen includes a sugar moiety. Tandem mass spectrometry analysis of the antigen revealed that the mAb recognizes sperm acrosin. When sodium dodecyl sulfate-solubilized PVM immobilized on a polyvinylidene difluoride membrane was incubated with sperm plasma membrane lysates, the sperm acrosin was detected on the PVM immobilized on the membrane, indicating that the sperm acrosin interacts with the components of PVM. Indeed, the mAb effectively inhibited the binding of acrosome-intact sperm to the PVM. These results indicate that the 45 kDa sperm acrosin is involved in the binding of sperm to the PVM in fertilization of Japanese quail.

scholarly journals Examination of the Anaerobic Growth ofCampylobacter concisusStrains

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Hoyul Lee ◽  
Rena Ma ◽  
Michael C. Grimm ◽  
Stephen M. Riordan ◽  
Ruiting Lan ◽  
...  
Keyword(s):  
Optimal Growth ◽  
Anaerobic Growth ◽  
Mass Spectrometry Analysis ◽  
Atmospheric Conditions ◽  
Anaerobic Conditions ◽  
Gas Generation ◽  
Spectrometry Analysis ◽  
Virulence Proteins ◽  
Oral Bacterium ◽  
Microaerobic Conditions

Campylobacter concisusis an oral bacterium that is associated with intestinal diseases.C. concisuswas previously described as a bacterium that requires H2-enriched microaerobic conditions for growth. The level of H2in the oral cavity is extremely low, suggesting thatC. concisusis unlikely to have a microaerobic growth there. In this study, the anaerobic growth ofC. concisuswas investigated. The growth of fifty-seven oralC. concisusstrains and six entericC. concisusstrains under various atmospheric conditions including anaerobic conditions with and without H2was examined. The atmospheric conditions were generated using commercially available gas-generation systems.C. concisusputative virulence proteins were identified using mass spectrometry analysis. Under anaerobic conditions, 92% of the oralC. concisusstrains (52/57) and all six enteric strains grew without the presence of H2and the presence of H2greatly increasedC. concisusgrowth. An oralC. concisusstrain was found to express a number of putative virulence proteins and the expression levels of these proteins were not affected by H2. The levels of H2appeared to affect the optimal growth ofC. concisus. This study provides useful information in understanding the natural colonization site and pathogenicity ofC. concisus.

scholarly journals Microdomain protein Nce102 is a local sensor of plasma membrane sphingolipid balance

2021 ◽  
Author(s):  
Jakub Zahumensky ◽  
Caroline Mota Fernandes ◽  
Petra Vesela ◽  
Maurizio Del Poeta ◽  
James Bernard Konopka ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Physiological Role ◽  
Building Blocks ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Signalling Molecules ◽  
Human Fungal Pathogen ◽  
Sphingolipid Biosynthesis ◽  
Complex Sphingolipids

Sphingolipids are essential building blocks of eukaryotic membranes and important signalling molecules, tightly regulated in response to environmental and physiological inputs. Mechanism of sphingolipid level perception at the plasma membrane remains unclear. In Saccharomyces cerevisiae, Nce102 protein has been proposed to function as sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that production of Nce102 increases following sphingolipid biosynthesis inhibition and Nce102 is internalized when excess sphingolipid precursors are supplied. This suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. Physiological role of Nce102 in regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of complex sphingolipids and long-chain bases in nce102? deletion mutant. Nce102 behaves analogously in human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species.

scholarly journals Physiological Adaptation of Desulfitobacterium hafniense Strain TCE1 to Tetrachloroethene Respiration

2011 ◽  
Vol 77 (11) ◽  
pp. 3853-3859 ◽  
Author(s):  
Laure Prat ◽  
Julien Maillard ◽  
Régis Grimaud ◽  
Christof Holliger
Keyword(s):  
Electron Acceptor ◽  
Growth Conditions ◽  
Mass Spectrometry Analysis ◽  
Physiological Adaptation ◽  
Terminal Electron Acceptor ◽  
Organohalide Respiration ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Metabolic Versatility ◽  
Desulfitobacterium Hafniense

ABSTRACTDesulfitobacteriumspp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells ofDesulfitobacterium hafniensestrain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the Wood-Ljungdahl pathway and electron transfer flavoproteins. These proteomic results suggest thatD. hafniensestrain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.

scholarly journals Transcriptional Regulation of the Two Sterol Esterification Genes in the Yeast Saccharomyces cerevisiae

2001 ◽  
Vol 183 (17) ◽  
pp. 4950-4957 ◽  
Author(s):  
Kristen Jensen-Pergakes ◽  
Zhongmin Guo ◽  
Mara Giattina ◽  
Stephen L. Sturley ◽  
Martin Bard
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Physiological Role ◽  
Growth Conditions ◽  
Product Inhibition ◽  
Anaerobic Growth ◽  
Primary Role ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sterol Esterification

ABSTRACT Saccharomyces cerevisiae transcribes two genes,ARE1 and ARE2, that contribute disproportionately to the esterification of sterols. Are2p is the major enzyme isoform in a wild-type cell growing aerobically. This likely results from a combination of differential transcription initiation and transcript stability. By using ARE1 andARE2 promoter fusions to lacZ reporters, we demonstrated that transcriptional initiation from theARE1 promoter is significantly reduced compared to that from the ARE2 promoter. Furthermore, the half-life of the ARE2 mRNA is approximately 12 times as long as that of the ARE1 transcript. We present evidence that the primary role of the minor sterol esterification isoform encoded byARE1 is to esterify sterol intermediates, whereas the role of the ARE2 enzyme is to esterify ergosterol, the end product of the pathway. Accordingly, the ARE1promoter is upregulated in strains that accumulate ergosterol precursors. Furthermore, ARE1 and ARE2are oppositely regulated by heme. Under heme-deficient growth conditions, ARE1 was upregulated fivefold whileARE2 was down-regulated. ARE2 requires the HAP1 transcription factor for optimal expression, and both ARE genes are derepressed in arox1 (repressor of oxygen) mutant genetic background. We further report that the ARE genes are not subject to end product inhibition; neither ARE1 nor ARE2transcription is altered in an are mutant background, nor does overexpression of either ARE gene alter the response of the ARE-lacZ reporter constructs. Our observations are consistent with an important physiological role for Are1p during anaerobic growth when heme is limiting and sterol precursors may accumulate. Conversely, Are2p is optimally required during aerobiosis when ergosterol is plentiful.

scholarly journals Quantitative Proteomic Analysis of Biogenesis-Based Classification for Extracellular Vesicles

Proteomes ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 33
Author(s):  
Linwen Zhang ◽  
Jeremie Parot ◽  
Vincent A. Hackley ◽  
Illarion V. Turko
Keyword(s):  
Mass Spectrometry ◽  
Plasma Membrane ◽  
Extracellular Vesicles ◽  
Specific Protein ◽  
Mass Spectrometry Analysis ◽  
Protein Markers ◽  
Spectrometry Analysis ◽  
Endosomal Membrane ◽  
Size Limitation ◽  
Endosomal Membranes

Extracellular vesicles (EVs) are traditionally divided into two major groups: (i) large vesicles originating from plasma membrane and called microvesicles, and (ii) small vesicles originating from the endoplasmic membrane and called exosomes. However, it is increasingly clear that the actual composition of a particular EV preparation cannot be adequately described with these two simple terms and is much more complex. Since the cell membrane origin of EVs predetermines their biological functions, the understanding of EV biogenesis is important for accurate interpretation of observed results. In the present study, we propose to take advantage of selective expression of some proteins in plasma or endosomal membranes and to use these proteins as plasma membrane-specific or endosomal membrane-specific markers. We have demonstrated that a quantitative mass spectrometry analysis allows simultaneous measurement of plasma membrane-specific and endosomal membrane-specific proteins in microvesicles and exosomes obtained after differential ultracentrifugation. Before mass spectrometry analysis, we also used sonicated platelets as a model of mixed EVs and multidetector asymmetrical-flow field-flow fractionation as an analytical method to verify a possible cross contamination of obtained microvesicles and exosomes. Based on the quantitative appearance of membrane-specific protein markers in EV preparations from human plasma and from human ARPE-19 cell medium, we concluded that there is no actual size limitation and both microvesicles and exosomes can be represented by large and small vesicles.

scholarly journals In Nasopharyngeal Carcinoma Cells, Epstein-Barr Virus LMP1 Interacts with Galectin 9 in Membrane Raft Elements Resistant to Simvastatin

2005 ◽  
Vol 79 (21) ◽  
pp. 13326-13337 ◽  
Author(s):  
Catherine Pioche-Durieu ◽  
Cécile Keryer ◽  
Sylvie Souquère ◽  
Jacques Bosq ◽  
Wolfgang Faigle ◽  
...  
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Mass Spectrometry Analysis ◽  
Membrane Rafts ◽  
Independent Manner ◽  
Mutant Proteins ◽  
Spectrometry Analysis ◽  
Barr Virus ◽  
Cell Extracts ◽  
Epstein Barr

ABSTRACT Nasopharyngeal carcinomas (NPC) are etiologically related to the Epstein-Barr virus (EBV), and malignant NPC cells have consistent although heterogeneous expression of the EBV latent membrane protein 1 (LMP1). LMP1 trafficking and signaling require its incorporation into membrane rafts. Conversely, raft environment is likely to modulate LMP1 activity. In order to investigate NPC-specific raft partners of LMP1, rafts derived from the C15 NPC xenograft were submitted to preparative immunoprecipitation of LMP1 combined with mass spectrometry analysis of coimmunoprecipitated proteins. Through this procedure, galectin 9, a beta-galactoside binding lectin and Hodgkin tumor antigen, was identified as a novel LMP1 partner. LMP1 interaction with galectin 9 was confirmed by coimmunoprecipitation and Western blotting in whole-cell extracts of NPC and EBV-transformed B cells (lymphoblastoid cell lines [LCLs]). Using mutant proteins expressed in HeLa cells, LMP1 was shown to bind galectin 9 in a TRAF3-independent manner. Galectin 9 is abundant in NPC biopsies as well as in LCLs, whereas it is absent in Burkitt lymphoma cells. In subsequent experiments, NPC cells were treated with Simvastatin, a drug reported to dissociate LMP1 from membrane rafts in EBV-transformed B cells. We found no significant effects of Simvastatin on the distribution of LMP1 and galectin 9 in NPC cell rafts. However, Simvastatin was highly cytotoxic for NPC cells, regardless of the presence or absence of LMP1. This suggests that Simvastatin is a potentially useful agent for the treatment of NPCs although it has distinct mechanisms of action in NPC and LCL cells.

scholarly journals Localization and Developmental Regulation of a Dispersed Gene Family 1 Protein in Trypanosoma cruzi

2009 ◽  
Vol 78 (1) ◽  
pp. 231-240 ◽  
Author(s):  
Noelia Lander ◽  
Carolina Bernal ◽  
Nardy Diez ◽  
Néstor Añez ◽  
Roberto Docampo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Gene Family ◽  
Culture Medium ◽  
Close Contact ◽  
Developmental Regulation ◽  
Developmental Expression ◽  
Mass Spectrometry Analysis ◽  
Western Blots ◽  
Spectrometry Analysis ◽  
Amastigote Stage

ABSTRACT The dispersed gene family 1 (DGF-1) is the fifth largest gene family in the T rypanosoma cruzi genome, with over 500 members (11). Many of the predicted DGF-1 protein products have several transmembrane domains and N-glycosylation and phosphorylation sites and were thought to localize in the plasma membrane. Here, we report that affinity-purified antibodies against a region of one of these proteins (DGF-1.2) localized it intracellularly in different stages of the parasite. DGF-1.2 is more abundant in the amastigote stage than in trypomastigotes and epimastigotes, as detected by immunofluorescence and Western blot analyses. The protein changed localization during intracellular or extracellular differentiation from the trypomastigote to the amastigote stage, where it finally localized to small bodies in close contact with the inner side of the amastigote plasma membrane. DGF-1.2 did not colocalize with markers of other subcellular organelles, such as acidocalcisomes, glycosomes, reservosomes, lipid droplets, or endocytic vesicles. During extracellular differentiation, the protein was detected in the culture medium from 0 to 22 h, peaking at 14 h. The presence of DGF-1.2 in the differentiation culture medium was confirmed by mass spectrometry analysis. Finally, when epimastigotes were subjected to starvation, there was a decrease in the labeling of the cells and, in Western blots, the appearance of bands of lower molecular mass, suggesting its cleavage. These results represent the first report of direct immunodetection and developmental expression and secretion of a DGF-1 protein.

scholarly journals Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for anaerobic growth

2021 ◽  
Author(s):  
Wijb J.C. Dekker ◽  
Raúl Ortiz-Merino ◽  
Astrid Kaljouw ◽  
Frank Willem Wiering ◽  
Christiaan Mooiman ◽  
...  
Keyword(s):  
Kluyveromyces Marxianus ◽  
Large Scale ◽  
Industrial Applications ◽  
Economic Benefits ◽  
Anaerobic Growth ◽  
Uptake Mechanism ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sterol Uptake ◽  
Key Factor ◽  
Independent Synthesis

Current large-scale, anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Response of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for anaerobic industrial applications.

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