Fig 1.
Ty1 mobility and protein levels in cells repopulated with Ty1 elements.
A Ty1-less S. paradoxus strain repopulated with increasing copies of genomic Ty1-H3 elements [YEM514 (1 Ty1), YEM568 (9 Ty1s), YEM570 (15 Ty1s), YEM572 (22 Ty1s), and YEM515 (38 Ty1s)] and containing pGTy1his3-AI/URA3/CEN (pBDG606) were analyzed for Ty1his3-AI mobility and protein levels. All strains were deleted for SPT3, which blocks Ty1 mRNA, but not Ty1i RNA, expression from genomic loci. (A) A quantitative Ty1his3-AI mobility assay was performed with galactose-induced cells. Numbers represent Ty1his3-AI mobility events per cell and bars represent standard deviations. Mobility assays were repeated at least three times and representative results are shown. (B) Cells were grown for 24 h under repressing (glucose) or inducing (galactose) conditions for pGTy1his3-AI expression. 1 and 38 Ty1 copy strains containing an empty vector were used as controls. TCA-precipitated protein extracts were immunoblotted with p18 antibody, which recognizes full length immature and mature Gag (p49/p45) and p22/p18, and Hts1 antibody, which served as a loading control. Separation of Gag-p49 and p45 does not occur under these electrophoresis conditions (see Materials and Methods) and thus are bracketed and collectively labeled as Gag-p49/p45. (C) Cells were induced for pGTy1 expression for 24 h and protein extracts were immunoblotted with IN, RT, or Hts1 antibody. (D) The 38 Ty1 copy strain containing WT or PR-defective (SacI linker–1702 [47]) pGTy1/2μ plasmids were induced with galactose for 24 h. TCA-precipitated extracts were immunoblotted with p18 antibody.
Fig 2.
Ty1 GAG mutations are specific and partially resistant to CNC.
S. paradoxus repopulated with 38 (YEM515; A) or 1 (YEM514; B) genomic copies of Ty1-H3 and containing wild type or CNCR pGTy1his3-AI plasmids were analyzed for Ty1his3-AI mobility. Ty1his3-AI mobility for each mutant was normalized to wild type and fold changes are represented. Raw data can be found in S4 Table. (C) Percent CNC recovery was calculated by dividing Ty1his3-AI mobility in the 38 Ty1 copy strain by mobility in the 1 copy strain. Recoveries above 10% are shown.
Table 1.
Genomic CNC-resistant Ty1his3-AI mobility.
Table 2.
Ty1his3-AI mobility in CNC-resistant Ty1 repopulated strains.
Fig 3.
Predicted helical structure of Ty1 Gag and location of CNCR substitutions.
(A) Ty1 Gag sequence (UniProt P08405) was analyzed for secondary structure prediction and domain identification. Predicted helical regions are represented by gray boxes below and labeled 1–9. Two Pfam domains, TYA (PF01021, residues 17–114) and UBN2 (PF14223, residues 245–356), are present in Ty1 Gag. Several CNCR residues clustered between residues 170–220, which we define as the CNCR domain. Other features include the Ty1 p22 protein (mapped underneath Ty1 Gag), the PR cleavage sites in Ty1 Gag and p22 proteins (H402/N403 in Gag, scissors) [54], a region that exhibits nucleic acid chaperone (NAC) activity (black bar) [28], and important Gag amino acids (black circles): a highly conserved tryptophan residue in Ty1/copia (W184) [53], and hydrophobic residues previously shown to be important for assembly (IM248/249, L252, LF339/340, and I343) [55]. (B, C) An alignment of yeast Ty1-like Gag sequences was generated with ClustalW and visualized with Jalview using the ClustalX color scheme. The CNCR (B) and UBN2 (C) domains were chosen for display. Species, UniProt annotations, and element type are listed to the left and include Ty1 and Ty2 elements from S. cerevisiae (Scer), Ty1-like elements from S. kudriavzevii (Skud) [3, 56] and the Tsk1 element from Lachancea kluyveri (Lklu) [57]. Ty1-H3 and Ty2-917 were included, however, these elements were isolated as spontaneous retrotransposition events and are unique from known genomic elements [58, 59]. Ty1 Gag residue coordinates and CNCR substitutions (above), predicted helical regions (gray boxes, below) and important residues (below) from (A) are labeled.
Fig 4.
Alterations in Gag helical domains disrupt transposition and Ty1 protein cleavage.
S. paradoxus repopulated with 1 (YEM514) or 38 (YEM515) genomic copies of Ty1-H3 and containing wild type or helix mutant pGTy1his3-AI plasmids were analyzed for Ty1his3-AI mobility using a qualitative plate assay (A) and for Ty1 protein levels (B). (A) Cells grown on SC-Ura were induced for pGTy1his3-AI expression and retrotransposition by replica plating to SC-Ura + 2% galactose and incubating at 22°C for 2 days. Cell patches were replicated to SC-Ura-His and grown at 30°C until His+ papillae appeared. Each His+ colony contains at least one Ty1HIS3 insertion. (B) Cells were induced in liquid SC-Ura + 2% galactose medium for 24 h and TCA-extracted proteins were immunoblotted with p18, RT, and Hts1 antibodies. (C) Cell extracts from 1 Ty1 copy strain expressing helix mutant pGTy1his3-AI plasmids were centrifuged through a 7–47% continuous sucrose gradient. Equal volumes of each fraction were immunoblotted with TY antibody, which recognizes full length Gag (p49/p45).
Fig 5.
CNCR mutations N183D, K186Q, I201T, and A273V alter Ty1 protein levels.
S. paradoxus repopulated with 38 genomic copies of Ty1-H3 (YEM515) containing wild type or CNCR pGTy1his3-AI plasmids were analyzed for Ty1 protein levels. Cells were induced in liquid SC-Ura + 2% galactose for 24 h. (A) TCA-extracted proteins were immunoblotted with p18 and Hts1 antibodies. (B) Whole cell extracts were immunoblotted with RT and Hts1 antibodies.
Fig 6.
I201T CNCR VLPs have increased IN and RT protein levels and less p18.
VLPs were isolated from S. paradoxus repopulated with 38 genomic copies of Ty1-H3 (YEM515) containing wild type or CNCR pGTy1his3-AI-I201T that were induced for expression. Equal amounts (2 μg) of VLPs were immunoblotted with (A) VLP, (B) RT, and (C) IN antibodies. (D) Dilutions of WT and I201T VLPs were analyzed with p18 antibody. (E) Northern blotting of total RNA and VLP RNA was performed using Ty1 (nucleotides 335–550) and ACT1 32P-labeled riboprobes. Note that ACT1 transcripts are not detected in purified VLPs.
Table 3.
CNC-resistant pGTy1his3-AI mobility when co-expressed with p22-V5.
Fig 7.
I201T CNCR VLP assembly excludes p22-V5.
Protein extracts (input) from S. paradoxus strains (DG3508) co-expressing WT (pBDG1534) or pGTy1his3-AI-I201T (pBJM24) and an empty vector (pRS416; A, B) or p22-V5 (pBJM93; C-F) were centrifuged through a 7–47% continuous sucrose gradient. Equal volumes of each fraction were immunoblotted with p18 (A-C, E) and V5 antibodies (D, F).