1. Phosphorylation Regulates Integration of the Yeast Ty5 Retrotransposon into Heterochromatin 
Junbiao Dai, Weiwu Xie, Troy L. Brady, Jiquan Gao, Daniel F. Voytas 
Molecular Cell 
Volume 27, Issue 2, Pages (July 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Posttranslational Modification of the Ty5 Targeting Domain
(A) In vitro interaction between Sir4C and TD. Sir4C was expressed and labeled with 35S-methionine by coupled transcription and translation. GBD-TD (LDSSPPNTS) and GBD-td (LDLSPPNTS) were expressed in yeast and immunoaffinity purified with anti-GBD agarose beads. GST-TD and GST-td were expressed in E. coli and purified with glutathione agarose beads. As indicated by the top row of lanes, Sir4C interacts with TD purified from yeast, but not from E. coli. The interaction between Sir4C and TD is abrogated when GBD-TD immunopurified from yeast is treated with λ protein phosphatase before mixing with Sir4C. For each experiment, the bottom row of lanes indicates the amount of input protein used to test in vitro interactions. For GST and the GST fusion proteins, a Coomassie-stained gel of the input proteins is shown. For GBD and the GBD-TD fusions, the amount of input protein was assessed by immunoblotting using an anti-GBD antibody.
(B) Amino acid substitutions in TD suggest serine phosphorylation in a tethered silencing assay. Silencing is established when GBD-TD is tethered to a silencing-impaired HMR locus with UASg sites. Serial, 10-fold dilutions of cells were plated onto nonselective (SC-Ura) or selective (SC-Ura-Trp) media to measure silencing of the TRP1 reporter gene at HMR. Amino acid sequences of TD carried by the fusion proteins are shown on the left of the figure. Mutated residues are in bold. At the bottom of the figure is an immunoblot performed with an anti-GBD antibody to show that expression of each fusion protein was equivalent.
(C) Target specificity of various Ty5 mutants as measured by the plasmid-based targeting assay (Gai and Voytas, 1998). The y axis indicates the percentage of target plasmids with an HMR locus that acquired a Ty5 insertion. Average values and standard deviations from three independent experiments are shown.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
TD Is Phosphorylated In Vivo, and Phosphoryation Is Required for the Interaction with Sir4C
(A) Two-hybrid assays measure interactions between Sir4C and His6-tagged TD. The addition of His6 at either the N or C terminus of TD does not interfere with TD's ability to interact with Sir4C. Nonselective medium is SC-Leu-Trp; selective medium is SC-Leu-Trp-His + 1 mM 3-AT.
(B) Deconvoluted mass spectrum of His6-tagged TD. Values above each peak are the observed mass of the most abundant isotopic species. The observed mass difference between unphosphorylated TD and monophosphorylated TD closely corresponds to the calculated monoisotopic mass of HPO3, which is Da.
(C) Ion fragmentations from tandem MS of the ion fragment corresponding to monophosphorylated TD (m/z in [B]). More than 70% of the observed peaks could be assigned when S1095 was considered to be the phosphorylation site. b∗ and b∗∗ are produced by the loss of HPO3, and one or two H2O molecules from b ions. bΔ and yΔ ions are produced by the loss of one H2O from b ions and y ions, respectively.
(D) SPR demonstrates an interaction between phosphorylated TD and Sir4C. A significant association is only detected when the second serine within TD is phosphorylated (pS1095).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3 The Phosphorylation State of TD Is Regulated by Stress
(A) Specificity of the phospho-TD antibody. The phospho-TD antibody reacts strongly with GST-TD expressed and purified from yeast, whereas little or no reaction is observed with GST-TD purified from E. coli.
(B) TD phosphorylation levels decrease upon starvation for amino acids, nitrogen, or carbon. GST-TD phosphorylation was monitored using an antibody specific for phospho-TD after exposure of yeast cells to a variety of stress and growth conditions (upper panel) (see also the Experimental Procedures for a more detailed description of the stress regimes). An anti-GST antibody was used to assess overall levels of GST-TD protein expression (lower panel). Numbers above each lane indicate the hours after induction of GST-TD expression that the cells were harvested.
(C) Proteins isolated from 3 hr time points for the no stress control and the nutrient deprivation treatments were normalized with respect to GST using the anti-GST antibody. As a control for the specificity of the phospho-TD antibody, 50 ng of GST-TD protein that had been previously isolated from yeast or E. coli was included on the filter.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions