Download presentation
Presentation is loading. Please wait.
1
Volume 26, Issue 1, Pages 15-25 (April 2007)
Mapping Protein-Protein Interactions for the Yeast ABC Transporter Ycf1p by Integrated Split-Ubiquitin Membrane Yeast Two-Hybrid Analysis Christian M. Paumi, Javier Menendez, Anthony Arnoldo, Kim Engels, Kavitha Ravee Iyer, Safia Thaminy, Oleg Georgiev, Yves Barral, Susan Michaelis, Igor Stagljar Molecular Cell Volume 26, Issue 1, Pages (April 2007) DOI: /j.molcel Copyright © 2007 Elsevier Inc. Terms and Conditions
2
Figure 1 Strategy for Endogenous Cub-YFP-TF Tagging of a Yeast ORF Encoding an Integral Membrane Protein The oligonucleotides X and Y, corresponding to the indicated regions of the “bait”-encoding ORF, are used to PCR amplify a fragment from the pCYT-L3 plasmid containing the Cub-YFP-TF (CYT) cassette and a KanMX resistance marker gene (top panel). This PCR product is transformed into the iMYTH-specific yeast reporter strain THY AP4, and the PCR product integrates via homologous recombination to generate the C-terminally CYT-tagged integral membrane bait protein, shown in the bottom panel. The oligonucleotides Z and K are used to check the correct tagging of the ORF by colony PCR and sequencing (middle panel). Note that the KanMX gene is flanked by two loxP loci that could be used to pop out the KanMX cassette from the endogenously tagged yeast ORF by expressing the Cre Recombinase from a plasmid. Molecular Cell , 15-25DOI: ( /j.molcel ) Copyright © 2007 Elsevier Inc. Terms and Conditions
3
Figure 2 Application of iMYTH System to Ycf1p
(A) The proposed topology of the YCF1-CYT (C, Cub; Y, YFP; and T, the transcription factor [TF]) bait protein used in this study. The three membrane-spanning domains (MSD0, MSD1, and MSD2) and two nucleotide binding domains (NBD1 and NBD2) of Ycf1p are shown (not to scale). The CYT tag was fused to the C terminus of Ycf1p by the strategy described in Figure 1. (B) Endogenously tagged YCF1-CYT is functionally active. Cadmium sensitivity was tested by the spot dilution plate assay as described in the Supplemental Data. (C) Bait-dependency test showing the newly identified Ycf1p-interacting proteins (Ycf1p-IPs) identified by the modified iMYTH approach. The isolated interacting prey-NubG plasmids were reintroduced into the THY AP4 strain expressing either endogenously tagged YCF1-CYT or an unrelated vacuolar membrane protein VPS55-CYT. Specific activation of the reporter genes in the YCF1-CYT bait strain, but not in the unrelated VPS55-CYT bait strain, shows that the interacting preys are Ycf1p-dependent interactors. β-galactosidase assay that quantifies the strength of interactions between Ycf1p and Ycf1p-IPs is shown on the right. The error bars show the standard deviations determined from triplicates. (D) The Ycf1p interactome identified by iMYTH assay. The six Ycf1p-IPs connected with black lines are shown as color-coded circles according to the gene ontology annotation of biological process and their molecular function. The graph was produced by using the Osprey software ( Molecular Cell , 15-25DOI: ( /j.molcel ) Copyright © 2007 Elsevier Inc. Terms and Conditions
4
Figure 3 Cadmium and Arsenite Sensitivity of Mutant Strains Deleted for Ycf1p Interactors (A) Growth tests in liquid media to examine the cadmium and arsenite sensitivity of mutant strains deleted for Ycf1p interactors were performed as described in the Supplemental Data. Strains are WT (SM4460) and deletion mutants (SM5270-SM5273 and SM5275-SM5276). Individual columns represent the mean of three experiments (n = 3), and error bars represent the standard deviation from the mean. (B) The GFP fluorescence pattern of Ycf1p-GFP is shown for strains deleted for each of the Ycf1p interactors. The vacuole is an indented structure in the DIC images. Strains from left to right are SM4460, SM5270-SM5273, SM5275, and SM5276 all transformed with pSM1753 [2μ YCF1-GFP]. Molecular Cell , 15-25DOI: ( /j.molcel ) Copyright © 2007 Elsevier Inc. Terms and Conditions
5
Figure 4 Probing the Interaction between Ycf1p and Tus1p
(A) Ycf1p and Tus1p form a complex as indicated by coimmunoprecipitation experiment. HA-tagged TUS1 in pAB117 was transformed into THY AP4 expressing no other tagged proteins and into AP4 expressing either endogenously tagged YCF1-CYT or YBT1-CYT. Protein extracts prepared from these strains were immunoprecipitated (IP) with antibodies that recognize either epitope tag (anti-HA in the case of Tus1p or anti-LexA in the case of YCF1-CYT) as indicated. Immunoprecipitated proteins were resolved by SDS-PAGE (10% gel) and analyzed by western blotting using anti-HA or anti-LexA antibodies. Lanes 1, 2, and 3 are the inputs for the extracts used in the coimmunoprecipitation experiments. (B) Mapping the region of Tus1p that interacts with Ycf1p. The yeast reporter strain THY AP4 expressing YCF1-CYT from the chromosome was transformed with prey plasmids encoding the indicated Tus1p fragments fused to NubG. Three independent colonies were restreaked on SD-W and SD-WAH selective plates containing X-gal in the medium (right). Blue coloration of colonies is a marker of interaction. Full-length (FL) Tus1p is also shown, with a DH domain (blue, aa 472–656), a PH domain (yellow, aa 716–879), and a structurally unknown citron-like (CNH) domain (red, aa 939–1287). Molecular Cell , 15-25DOI: ( /j.molcel ) Copyright © 2007 Elsevier Inc. Terms and Conditions
6
Figure 5 Tus1p Stimulates Ycf1p-Mediated Transport In Vitro and Acts through Rho1p (A) In vitro transport of [H3]E2β17G into vessiculated vacuoles was measured as described in the Experimental Procedures in the presence of ATP or GTP either with or without addition of cytosolic extract, as indicated. Vacuoles are prepared from YCF1 (SM4460) or Δycf1 (SM5270) strains, indicated by the black and gray bars, respectively. (B) In vitro Ycf1p-dependent transport of [H3]E2β17G into vessiculated vacuoles prepared from a WT (YCF1+) strain was measured in the presence of GTP. Where indicated, cytosolic extract derived from WT or mutant strains was added to the transport reaction. Cytosol was prepared from the following strains: SM4460 (column 2), SM5272 (column 3), and SM5291 (column 4). (C) Ycf1p-dependent transport of [H3]E2β17G was measured as in (B), except that cytosol was replaced by adding recombinant His-Tus1p (+His-Tus1p) or the analogous Ni+2 column elution fraction prepared from E. coli not expressing the His-Tus1p construct (−His-Tus1p). (D) In vitro transport into vacuoles from strains harboring mutations in the rho genes. In vitro Ycf1p-dependent transport of [H3]E2β17G was measured with vacuoles derived from strains deleted for Δrho2, Δrho3, Δrho4, and Δrho5 or containing temperature-sensitive rho1ts. For the latter, a rho1Δ strain that contains plasmids bearing rho1-2ts or rho1-11ts was used because RHO1 is an essential gene. Ycf1p transport activity was measured in the presence of GTP with or without recombinant His-Tus1p (right and left bars, respectively). Strains are SM4460 (column 1), SM5270 (column 2), SM5293-SM5294 (columns 3 and 4), and SM5287-SM5290 (columns 5–8). For all experiments shown above, individual columns represent the mean of three experiments (n = 3) and error bars represent the standard deviation from the mean. Molecular Cell , 15-25DOI: ( /j.molcel ) Copyright © 2007 Elsevier Inc. Terms and Conditions
7
Figure 6 A Working Model Depicting the Proposed Interactions between Ycf1p, Tus1p, and Rho1p that Result in the Stimulation of Ycf1p-Dependent Transport Our working model, based on data presented here, is that Rho1p and Ycf1p are localized to the vacuole membrane; however, Rho1p has no effect on basal Ycf1p-transport activity (pale arrow). When Tus1p is recruited from the cytosol, it binds Ycf1p through its CNH and Rho1p via its DH domain, thereby bringing Ycf1p and Rho1p in close proximity and bridging their interaction. At this time, Tus1p stimulates the exchange of GTP for GDP by Rho1p, yielding activated Rho1p-GTP. The activation of Rho1p results in the stimulation of Ycf1p activity (dark arrow) by an as of yet undefined mechanism. Molecular Cell , 15-25DOI: ( /j.molcel ) Copyright © 2007 Elsevier Inc. Terms and Conditions
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.