scholarly journals Structure and Function Analysis of CaMdr1p, a Major Facilitator Superfamily Antifungal Efflux Transporter Protein of Candida albicans: Identification of Amino Acid Residues Critical for Drug/H+ Transport

2007 ◽  
Vol 6 (3) ◽  
pp. 443-453 ◽  
Author(s):  
Ritu Pasrija ◽  
Dibyendu Banerjee ◽  
Rajendra Prasad
Keyword(s):  
Drug Resistance ◽  
Drug Transport ◽  
Fluorescent Protein ◽  
Function Analysis ◽  
Major Facilitator Superfamily ◽  
Site Directed Mutagenesis ◽  
Core Motif ◽  
Transporter Protein ◽  
First Category ◽  
Energy Inhibitors

ABSTRACT We have cloned and overexpressed multidrug transporter CaMdr1p as a green fluorescent protein-tagged protein to show its capability to extrude drug substrates. The drug extrusion was sensitive to pH and energy inhibitors and displayed selective substrate specificity. CaMdr1p has a unique and conserved antiporter motif, also called motif C [G(X6)G(X3)GP(X2)GP(X2)G], in its transmembrane segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site-directed mutagenesis highlighted the importance of the motif, as well as that of other residues of TMS 5, in drug transport. The mutant variants of TMS 5 were placed in four different categories. The first category had four residues, G244, G251, G255, and G259, which are part of the conserved motif C, and their substitution with alanine resulted in increased sensitivity to drugs and displayed impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine, resulted in more dramatic loss of drug resistance and efflux. Notwithstanding the location in the core motif, the second category included residues which are part of the motif, such as P260, and those which were not part of the motif, such as L245, W248, P256, and F262, whose substitution with alanine resulted in a severe loss of drug resistance and efflux. The third category included G263, which is a part of motif C, but unlike other conserved glycines, its replacement with alanine or leucine showed no change in the phenotype. The replacement of the remaining 11 residues of the fourth category did not result in any change. The putative helical wheel projection showed clustering of functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5.

scholarly journals Multidrug Transporters CaCdr1p and CaMdr1p of Candida albicans Display Different Lipid Specificities: both Ergosterol and Sphingolipids Are Essential for Targeting of CaCdr1p to Membrane Rafts

2007 ◽  
Vol 52 (2) ◽  
pp. 694-704 ◽  
Author(s):  
Ritu Pasrija ◽  
Sneh Lata Panwar ◽  
Rajendra Prasad
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Membrane Lipid ◽  
Major Facilitator Superfamily ◽  
Pathogenic Yeast ◽  
Atp Binding Cassette Transporter ◽  
Major Facilitator ◽  
Green Fluorescent

ABSTRACT In this study, we compared the effects of altered membrane lipid composition on the localization of two membrane drug transporters from different superfamilies of the pathogenic yeast Candida albicans. We demonstrated that in comparison to the major facilitator superfamily multidrug transporter CaMdr1p, ATP-binding cassette transporter CaCdr1p of C. albicans is preferentially localized within detergent-resistant membrane (DRM) microdomains called ‘rafts.’ Both CaCdr1p and CaMdr1p were overexpressed as green fluorescent protein (GFP)-tagged proteins in a heterologous host Saccharomyces cerevisiae, wherein either sphingolipid (Δsur4 or Δfen1 or Δipt1) or ergosterol (Δerg24 or Δerg6 or Δerg4) biosynthesis was compromised. CaCdr1p-GFP, when expressed in the above mutant backgrounds, was not correctly targeted to plasma membranes (PM), which also resulted in severely impaired drug resistance. In contrast, CaMdr1p-GFP displayed no sorting defect in the mutant background and remained properly surface localized and displayed no change in drug resistance. Our data clearly show that CaCdr1p is selectively recruited, over CaMdr1p, to the DRM microdomains of the yeast PM and that any imbalance in the raft lipid constituents results in missorting of CaCdr1p.

scholarly journals Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members inCandida glabrataDrug Resistance Phenotypes

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Bao G. Vu ◽  
W. Scott Moye-Rowley
Keyword(s):  
Drug Resistance ◽  
Membrane Proteins ◽  
Common Species ◽  
Major Facilitator Superfamily ◽  
Content Type ◽  
Transporter Proteins ◽  
Transporter Protein ◽  
Major Facilitator ◽  
Encoding Genes

ABSTRACTCandida glabratais the second most common species causing candidiasis.C. glabratacan also readily acquire resistance to azole drugs, complicating its treatment. Here we add to the collection of disruption markers to aid in genetic analysis of this yeast. This new construct is marked with a nourseothricin resistance cassette that produces an estrogen-activated form of Cre recombinase in a methionine-regulated manner. This allows eviction and reuse of this cassette in a facile manner. Using this new disruption marker, we have constructed a series of strains lacking different members of the major facilitator superfamily (MFS) of membrane transporter proteins. The presence of 15 MFS proteins that may contribute to drug resistance inC. glabrataplaced a premium on development of a marker that could easily be reused to construct multiple gene-disrupted strains. Employing this recyclable marker, we found that loss of the MFS transporter-encoding geneFLR1caused a dramatic increase in diamide resistance (as seen before), and deletion of two other MFS-encoding genes did not influence this phenotype. Interestingly, loss ofFLR1led to an increase in levels of oxidized glutathione, suggesting a possible molecular explanation for this enhanced oxidant sensitivity. We also found that while overproduction of the transcription factor Yap1 could suppress the fluconazole sensitivity caused by loss of the important ATP-binding cassette transporter protein Cdr1, this required the presence ofFLR1.IMPORTANCEExport of drugs is a problem for chemotherapy of infectious organisms. A class of membrane proteins called the major facilitator superfamily contains a large number of proteins that often elevate drug resistance when overproduced but do not impact this phenotype when the gene is removed. We wondered if this absence of a phenotype for a disruption allele might be due to the redundancy of this group of membrane proteins. We describe the production of an easy-to-use recyclable marker cassette that will allow construction of strains lacking multiple members of the MFS family of transporter proteins.

scholarly journals Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Adam Taylor ◽  
Xiang Liu ◽  
Ali Zaid ◽  
Lucas Y. H. Goh ◽  
Jody Hobson-Peters ◽  
...  
Keyword(s):  
Host Cell ◽  
Capsid Protein ◽  
Chikungunya Virus ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Vaccine Design ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Nucleolar Localization ◽  
Localization Sequence

ABSTRACTMosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of theTogaviridaefamily responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infectionin vitro. Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design.IMPORTANCECHIKV is a mosquito-borne pathogen capable of causing explosive epidemics of incapacitating joint pain affecting millions of people. After a series of major outbreaks over the last 10 years, CHIKV and its mosquito vectors have been able to expand their range extensively, now making CHIKV a human pathogen of global importance. With no licensed vaccine or antiviral therapy for the treatment of CHIKV disease, there is a growing need to understand the molecular determinants of viral pathogenesis. These studies identify a previously uncharacterized nucleolar localization sequence (NoLS) in CHIKV capsid protein, begin a functional analysis of site-directed mutants of the capsid protein NoLS, and examine the effect of the NoLS mutation on CHIKV pathogenesisin vivoand its potential to influence CHIKV vaccine design. A better understanding of the pathobiology of CHIKV disease will aid the development of effective therapeutic strategies.

scholarly journals Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS256Tempering of WalKR Expression

2013 ◽  
Vol 57 (7) ◽  
pp. 3240-3249 ◽  
Author(s):  
Christopher R. E. McEvoy ◽  
Brian Tsuji ◽  
Wei Gao ◽  
Torsten Seemann ◽  
Jessica L. Porter ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Infection Model ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Phenotype Analysis ◽  
Dosing Regimens ◽  
Mrsa Strains ◽  
Green Fluorescent

ABSTRACTVancomycin-intermediateStaphylococcus aureus(VISA) strains often arise by mutations in the essential two-component regulatorwalKR; however their impact onwalKRfunction has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptibleS. aureus(VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256insertions in thewalKR5′ untranslated region (5′ UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction inwalKRgene expression in the IS256mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256insertions had replaced two SigA-likewalKRpromoters with weaker, hybrid promoters. Removal of IS256reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation ofssaAbut no change in expression ofsakandsceDin both IS256mutants. Whole-genome sequencing of the two mutants revealed an additional IS256insertion withinagrCfor one mutant, and we confirmed that this mutation abolishedagrfunction. These data provide the first substantial analysis ofwalKRpromoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction inwalKRexpression; however, the mechanisms by which this occurs remain to be determined.

Rab22a affects the morphology and function of the endocytic pathway

2001 ◽  
Vol 114 (22) ◽  
pp. 4041-4049 ◽  
Author(s):  
Rosana Mesa ◽  
Cristina Salomón ◽  
Marcelo Roggero ◽  
Philip D. Stahl ◽  
Luis S. Mayorga
Keyword(s):  
Fluorescent Protein ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Rab Proteins ◽  
Rab Protein ◽  
Late Endosomes ◽  
And Function ◽  
Negative Mutant

Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.

scholarly journals Structure-function analysis of human transforming growth factor-α by site-directed mutagenesis

1992 ◽  
Vol 283 (1) ◽  
pp. 91-98 ◽  
Author(s):  
J A Feild ◽  
R H Reid ◽  
D J Rieman ◽  
T P Kline ◽  
G Sathe ◽  
...  
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Receptor Binding ◽  
Transcriptional Control ◽  
Transforming Growth Factor ◽  
Function Analysis ◽  
Protein A ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Transforming Growth Factor Α

Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.

scholarly journals Nuclear localization of the Epstein–Barr virus EBNA3B protein

2006 ◽  
Vol 87 (4) ◽  
pp. 789-793 ◽  
Author(s):  
Anita Burgess ◽  
Marion Buck ◽  
Kenia Krauer ◽  
Tom Sculley
Keyword(s):  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Nuclear Antigen ◽  
Nuclear Localization Signals ◽  
Barr Virus ◽  
Computer Analyses ◽  
Epstein Barr ◽  
Green Fluorescent

The Epstein–Barr virus nuclear antigen (EBNA) 3B is a hydrophilic, proline-rich, charged protein that is thought to be involved in transcriptional regulation and is targeted exclusively to the cell nucleus, where it localizes to discrete subnuclear granules. Co-localization studies utilizing a fusion protein between enhanced green fluorescent protein (EGFP) and EBNA3B with FLAG-tagged EBNA3A and EBNA3C proteins demonstrated that EBNA3B co-localized with both EBNA3A and EBNA3C in the nuclei of cells when overexpressed. Computer analyses identified four potential nuclear-localization signals (NLSs) in the EBNA3B amino acid sequence. By utilizing fusion proteins with EGFP, deletion constructs of EBNA3B and site-directed mutagenesis, three of the four NLSs (aa 160–166, 430–434 and 867–873) were shown to be functional in truncated forms of EBNA3B, whilst an additional NLS (aa 243–246) was identified within the N-terminal region of EBNA3B. Only two of the NLSs were found to be functional in the context of the full-length EBNA3B protein.

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