scholarly journals Characterization of Three Classes of Membrane Proteins Involved in Fungal Azole Resistance by Functional Hyperexpression in Saccharomyces cerevisiae

2007 ◽  
Vol 6 (7) ◽  
pp. 1150-1165 ◽  
Author(s):  
Erwin Lamping ◽  
Brian C. Monk ◽  
Kyoko Niimi ◽  
Ann R. Holmes ◽  
Sarah Tsao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Abc Transporters ◽  
Fluorescent Protein ◽  
Major Facilitator Superfamily ◽  
Cytochrome P450 Enzyme ◽  
Heterologous Proteins ◽  
Functional Protein ◽  
Eukaryotic Membrane Proteins

ABSTRACT The study of eukaryotic membrane proteins has been hampered by a paucity of systems that achieve consistent high-level functional protein expression. We report the use of a modified membrane protein hyperexpression system to characterize three classes of fungal membrane proteins (ABC transporters Pdr5p, CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p, and CneMdr1p, the major facilitator superfamily transporter CaMdr1p, and the cytochrome P450 enzyme CaErg11p) that contribute to the drug resistance phenotypes of five pathogenic fungi and to express human P glycoprotein (HsAbcb1p). The hyperexpression system consists of a set of plasmids that direct the stable integration of a single copy of the expression cassette at the chromosomal PDR5 locus of a modified host Saccharomyces cerevisiae strain, ADΔ. Overexpression of heterologous proteins at levels of up to 29% of plasma membrane protein was achieved. Membrane proteins were expressed with or without green fluorescent protein (GFP), monomeric red fluorescent protein, His, FLAG/His, Cys, or His/Cys tags. Most GFP-tagged proteins tested were correctly trafficked within the cell, and His-tagged proteins could be affinity purified. Kinetic analysis of ABC transporters indicated that the apparent K m value and the V max value of ATPase activities were not significantly affected by the addition of His tags. The efflux properties of seven fungal drug pumps were characterized by their substrate specificities and their unique patterns of inhibition by eight xenobiotics that chemosensitized S. cerevisiae strains overexpressing ABC drug pumps to fluconazole. The modified hyperexpression system has wide application for the study of eukaryotic membrane proteins and could also be used in the pharmaceutical industry for drug screening.

scholarly journals Retrieval of Resident Late-Golgi Membrane Proteins from the Prevacuolar Compartment of Saccharomyces cerevisiae Is Dependent on the Function of Grd19p

1998 ◽  
Vol 140 (3) ◽  
pp. 577-590 ◽  
Author(s):  
Wolfgang Voos ◽  
Tom H. Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Localization ◽  
Transport Processes ◽  
Carboxypeptidase Y ◽  
Golgi Membrane ◽  
Cytosolic Domain ◽  
Prevacuolar Compartment ◽  
Vacuolar Protein

The dynamic vesicle transport processes at the late-Golgi compartment of Saccharomyces cerevisiae (TGN) require dedicated mechanisms for correct localization of resident membrane proteins. In this study, we report the identification of a new gene, GRD19, involved in the localization of the model late-Golgi membrane protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and lumenal domains of the alkaline phosphatase [ALP]), which localizes to the yeast TGN. A grd19 null mutation causes rapid mislocalization of the late-Golgi membrane proteins A-ALP and Kex2p to the vacuole. In contrast to previously identified genes involved in late-Golgi membrane protein localization, grd19 mutations cause only minor effects on vacuolar protein sorting. The recycling of the carboxypeptidase Y sorting receptor, Vps10p, between the TGN and the prevacuolar compartment is largely unaffected in grd19Δ cells. Kinetic assays of A-ALP trafficking indicate that GRD19 is involved in the process of retrieval of A-ALP from the prevacuolar compartment. GRD19 encodes a small hydrophilic protein with a predominantly cytosolic distribution. In a yeast mutant that accumulates an exaggerated form of the prevacuolar compartment (vps27), Grd19p was observed to localize to this compartment. Using an in vitro binding assay, Grd19p was found to interact physically with the cytosolic domain of DPAP A. We conclude that Grd19p is a component of the retrieval machinery that functions by direct interaction with the cytosolic tails of certain TGN membrane proteins during the sorting/budding process at the prevacuolar compartment.

scholarly journals Resistance and Adaptation to Quinidine in Saccharomyces cerevisiae: Role of QDR1 (YIL120w), Encoding a Plasma Membrane Transporter of the Major Facilitator Superfamily Required for Multidrug Resistance

2001 ◽  
Vol 45 (5) ◽  
pp. 1528-1534 ◽  
Author(s):  
Patrı́cia A. Nunes ◽  
Sandra Tenreiro ◽  
Isabel Sá-Correia
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Multidrug Resistance ◽  
Fluorescent Protein ◽  
Resistance Mechanisms ◽  
Major Facilitator Superfamily ◽  
Yeast Cells ◽  
Detectable Effect ◽  
Protein Fusion ◽  
Major Facilitator

ABSTRACT As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded by Saccharomyces cerevisiae open reading frameYIL120w is a multidrug resistance determinant. Yil120wp was implicated in yeast resistance to ketoconazole and quinidine, but not to the stereoisomer quinine; the gene was thus named QDR1. Qdr1p was proved to alleviate the deleterious effects of quinidine, revealed by the loss of cell viability following sudden exposure of the unadapted yeast population to the drug, and to allow the earlier eventual resumption of exponential growth under quinidine stress. However, QDR1 gene expression had no detectable effect on the susceptibility of yeast cells previously adapted to quinidine. Fluorescence microscopy observation of the distribution of the Qdr1-green fluorescent protein fusion protein in living yeast cells indicated that Qdr1p is a plasma membrane protein. We also show experimental evidence indicating that yeast adaptation to growth with quinidine involves the induction of active expulsion of the drug from preloaded cells, despite the fact that this antiarrhythmic and antimalarial quinoline ring-containing drug is not present in the yeast natural environment. However, we were not able to prove that Qdr1p is directly implicated in this export. Results clearly suggest that there are other unidentified quinidine resistance mechanisms that can be used in the absence of QDR1.

scholarly journals Immunoisolaton of the Yeast Golgi Subcompartments and Characterization of a Novel Membrane Protein, Svp26, Discovered in the Sed5-Containing Compartments

2005 ◽  
Vol 25 (17) ◽  
pp. 7696-7710 ◽  
Author(s):  
Hironori Inadome ◽  
Yoichi Noda ◽  
Hiroyuki Adachi ◽  
Koji Yoda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Membrane Protein ◽  
Considerable Portion ◽  
Marker Proteins ◽  
Evolutionarily Conserved ◽  
Golgi Compartment

ABSTRACT The Golgi apparatus consists of a set of vesicular compartments which are distinguished by their marker proteins. These compartments are physically separated in the Saccharomyces cerevisiae cell. To characterize them extensively, we immunoisolated vesicles carrying either of the SNAREs Sed5 or Tlg2, the markers of the early and late Golgi compartments, respectively, and analyzed the membrane proteins. The composition of proteins was mostly consistent with the position of each compartment in the traffic. We found six uncharacterized but evolutionarily conserved proteins and named them Svp26 (Sed5 compartment vesicle protein of 26 kDa), Tvp38, Tvp23, Tvp18, Tvp15 (Tlg2 compartment vesicle proteins of 38, 23, 18, and 15 kDa), and Gvp36 (Golgi vesicle protein of 36 kDa). The localization of Svp26 in the early Golgi compartment was confirmed by microscopic and biochemical means. Immunoprecipitation indicated that Svp26 binds to itself and a Golgi mannosyltransferase, Ktr3. In the absence of Svp26, a considerable portion of Ktr3 was mislocalized in the endoplasmic reticulum. Our data suggest that Svp26 has a novel role in retention of a subset of membrane proteins in the early Golgi compartments.

scholarly journals GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

2008 ◽  
Vol 3 (5) ◽  
pp. 784-798 ◽  
Author(s):  
David Drew ◽  
Simon Newstead ◽  
Yo Sonoda ◽  
Hyun Kim ◽  
Gunnar von Heijne ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Proteins ◽  
Optimization Scheme ◽  
Eukaryotic Membrane Proteins

scholarly journals Deciphering the transcriptional changes in Escherichia coli strains C41(DE3) and C43(DE3) that makes them a superior choice for membrane protein production.

2020 ◽  
Author(s):  
Chaille Teresa Webb ◽  
Trevor Lithgow
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Parent Strain ◽  
Protein Production ◽  
Growth Conditions ◽  
Differentially Expressed ◽  
Structural Protein ◽  
Functional Protein ◽  
Genetic Changes ◽  
Strain Bl21

Abstract Background: The production of membrane proteins for functional and structural protein analysis remains a bottleneck in the continuing quest for understanding biological systems. For recombinant membrane proteins, the Walker strains C41(DE3) and C43(DE3) are a valuable tool because they are capable of producing levels of functional protein that would otherwise be toxic to the cell. At the genome level, amongst only a handful of genetic changes, mutations in the lacUV5 promoter region upstream from the bacteriophage T7 RNA polymerase gene distinguish these strains from BL21(DE3) but do not inform on how the strains have adapted for superior production of recombinant membrane proteins. Results: Comparative transcriptomic analyses revealed a moderate change in gene expression in C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) under standard growth conditions. However, under the conditions used for membrane protein production (with plasmid carriage and addition of IPTG), the differential response of C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) was striking. Over 2000 genes were differentially expressed in C41(DE3) with a two-fold change and false discover rate < 0.01 and 1700 genes differentially expressed in C43(DE3) compared to their parent strain BL21(DE3). Conclusion: These results illuminate the cellular adaptations occurring in the Walker strains to alleviate the toxic effects that can occur during membrane protein production, whilst providing changes in metabolism pathways required for membrane protein biogenesis. The BL21(DE3) derivatives strains C41(DE3) and C43(DE3), are adept to the process of membrane biogenesis in E. coli, making them superior to their parent strain for the production of membrane proteins and potentially other toxic proteins.

scholarly journals Deciphering the transcriptional changes in Escherichia coli strains C41(DE3) and C43(DE3) that makes them a superior choice for membrane protein production.

2020 ◽  
Author(s):  
Chaille Teresa Webb ◽  
Trevor Lithgow
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Parent Strain ◽  
Protein Production ◽  
Growth Conditions ◽  
Differentially Expressed ◽  
Structural Protein ◽  
Functional Protein ◽  
Genetic Changes ◽  
Strain Bl21

Abstract Background: The production of membrane proteins for functional and structural protein analysis remains a bottleneck in the continuing quest for understanding biological systems. For recombinant membrane proteins, the Walker strains C41(DE3) and C43(DE3) are a valuable tool because they are capable of producing levels of functional protein that would otherwise be toxic to the cell. At the genome level, amongst only a handful of genetic changes, mutations in the lac UV5 promoter region upstream from the bacteriophage T7 RNA polymerase gene distinguish these strains from BL21(DE3) but do not inform on how the strains have adapted for superior production of recombinant membrane proteins. Results: Comparative transcriptomic analyses revealed a moderate change in gene expression in C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) under standard growth conditions. However, under the conditions used for membrane protein production (with plasmid carriage and addition of IPTG), the differential response of C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) was striking. Over 2000 genes were differentially expressed in C41(DE3) with a two-fold change and false discover rate < 0.01 and 1700 genes differentially expressed in C43(DE3) compared to their parent strain BL21(DE3). Conclusion : These results illuminate the cellular adaptations occurring in the Walker strains in response to minimal genetic changes. These changes in the transcriptome may help alleviate the toxic effects that can occur and improve membrane protein production. The BL21(DE3) derivatives strains C41(DE3) and C43(DE3), are adept to the process of membrane biogenesis in E. coli , making them superior to their parent strain for the production of membrane proteins and potentially other toxic proteins.

scholarly journals Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae.

1992 ◽  
Vol 118 (3) ◽  
pp. 531-540 ◽  
Author(s):  
M Seeger ◽  
G S Payne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Cell Surface ◽  
Heavy Chain ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Golgi Membrane ◽  
Alpha Factor ◽  
Mutant Cells

The role of clathrin in retention of Golgi membrane proteins has been investigated. Prior work showed that a precursor form of the peptide mating pheromone alpha-factor is secreted by Saccharomyces cerevisiae cells which lack the clathrin heavy chain gene (CHC1). This defect can be accounted for by the observation that the Golgi membrane protein Kex2p, which initiates maturation of alpha-factor precursor, is mislocalized to the cell surface of mutant cells. We have examined the localization of two additional Golgi membrane proteins, dipeptidyl aminopeptidase A (DPAP A) and guanosine diphosphatase (GDPase) in clathrin-deficient yeast strains. Our findings indicate that DPAP A is aberrantly transported to the cell surface but GDPase is not. In mutant cells carrying a temperature-sensitive allele of CHC1 (chc1-ts), alpha-factor precursor appears in the culture medium within 15 min, and Kex2p and DPAP A reach the cell surface within 30 min, after imposing the nonpermissive temperature. In contrast to these immediate effects, a growth defect is apparent only after 2 h at the nonpermissive temperature. Also, sorting of the vacuolar membrane protein, alkaline phosphatase, is not affected in chc1-ts cells until 2 h after the temperature shift. A temperature-sensitive mutation which blocks a late stage of the secretory pathway, sec1, prevents the appearance of mislocalized Kex2p at the cell surface of chc1-ts cells. We propose that clathrin plays a direct role in the retention of specific proteins in the yeast Golgi apparatus, thereby preventing their transport to the cell surface.

scholarly journals Changes in membrane proteins associated with inhibition of the general amino acid permease of yeast (Saccharomyces cerevisiae)

1981 ◽  
Vol 196 (2) ◽  
pp. 531-536 ◽  
Author(s):  
J R Woodward ◽  
H L Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Wild Type ◽  
Amino Acid Permease ◽  
Yeast Saccharomyces Cerevisiae ◽  
General Amino Acid Permease ◽  
Wild Type Yeast

The general amino acid permease (‘Gap’) system of the wild-type yeast (Saccharomyces cerevisiae) strain Y185 is inhibited by the uptake and accumulation of its substrate amino acids. Surprisingly, this inhibition persists even after ‘pools’ of amino acids, accumulated initially, have returned to normal sizes. Recovery from this inhibition depends on a supply of energy and involves the synthesis of a membrane protein component of the Gap system.

scholarly journals Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

2010 ◽  
Vol 192 (23) ◽  
pp. 6160-6171 ◽  
Author(s):  
Cristina L. Marolda ◽  
Bo Li ◽  
Michael Lung ◽  
Mei Yang ◽  
Anna Hanuszkiewicz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
Major Facilitator Superfamily ◽  
Membrane Topology ◽  
Functional Protein ◽  
O Antigen ◽  
Cysteine Scanning Mutagenesis

ABSTRACT Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (WzxEcO157) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that WzxEcO157 consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.

scholarly journals Selection of Biophysical Methods for Characterisation of Membrane Proteins

2019 ◽  
Vol 20 (10) ◽  
pp. 2605 ◽  
Author(s):  
Tristan O. C. Kwan ◽  
Rosana Reis ◽  
Giuliano Siligardi ◽  
Rohanah Hussain ◽  
Harish Cheruvara ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Early Stage ◽  
Biological Membrane ◽  
Basic Research ◽  
Integral Membrane Protein ◽  
Integral Membrane Proteins ◽  
Functional Protein ◽  
Protein Research ◽  
Selection Of

Over the years, there have been many developments and advances in the field of integral membrane protein research. As important pharmaceutical targets, it is paramount to understand the mechanisms of action that govern their structure–function relationships. However, the study of integral membrane proteins is still incredibly challenging, mostly due to their low expression and instability once extracted from the native biological membrane. Nevertheless, milligrams of pure, stable, and functional protein are always required for biochemical and structural studies. Many modern biophysical tools are available today that provide critical information regarding to the characterisation and behaviour of integral membrane proteins in solution. These biophysical approaches play an important role in both basic research and in early-stage drug discovery processes. In this review, it is not our objective to present a comprehensive list of all existing biophysical methods, but a selection of the most useful and easily applied to basic integral membrane protein research.

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