Charakterisierung der Ubiquitinierung des Ku80-Proteins

Die an das humane Ku80-Protein angehefteten Ubiquitinketten sind hauptsächlich über die Lysine an den Positionen 33 und 63 miteinander verbunden.Eine Überexpression von BRCA1 bzw. RNF8 führt zu einer verstärkten Ubiquitinierung des humanen Ku80-Proteins.In einem Ku80-Fragment mit 168 Aminosäuren sind die Lysine an den Positionen 125/126/129 für die Ubiquitinierung verantwortlich.Die Reduktion der BRCA1 Menge mittels siRNA führt zu einer verringerten Bindung von humanem Ku80 an den DSB

RsfA and L14 and their interaction are conserved in bacteria and eukaryotic organelles.

<p>(A) Phylogenetic distribution of RsfA (Interpro entry IPR004394 [DUF143]) and ribosomal protein L14 (IPR000218) on the iTOL tree of life <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815-Letunic1" target="_blank">[59]</a>. Triangles indicate species in which the RsfA-L14 interaction was detected by binary detection assays (grey), co-purification with the LRS (white) or both (black). Known RsfA-L14/LRS interactions are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815.s005" target="_blank">Table S1</a>. (B) <i>T. pallidum</i> RsfA (TP0738) interacts strongly with L14 (TP0199) and very weakly with other proteins involved in translation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815-Titz1" target="_blank">[6]</a> in yeast-two-hybrid assays. C, control (with empty prey vector to measure self-activation of the bait). This interaction is also conserved in <i>E. coli</i> (C). (D, E) RsfA and L14 homologues from human and maize interact in pull down experiments. RsfA homologues were tagged with NusA-His₆ (N) and L14 homologues with maltose binding protein (M) (human mtRsfA = C7orf30, mitochondrial ribosomal protein L14 = L14_mt; maize RsfA = Iojap, maize chloroplastic L14 = RPL14); i = input samples, o = output samples. Constructs with the corresponding Interpro signatures and the range of cloned codons are illustrated on the right. (F) Human mitochondrial C7orf30 (mtRsfA) co-localizes with L14_mt exclusively into mitochondria as visualized by MitoTracker Green. Nuclei visualized by DRAQ5 (blue) and membranes by eCFP-membrane (cyan). Co-localization of both mtRsfA (C7orf30) and L14_mt in mitochondria is indicated in yellow. (G) Bi-molecular fluorescence complementation (BiFC) reveals the interaction of mtRsfA (C7orf30) and L14_mt in mitochondria. Overlay images represent DRAQ5 (blue), CFP-membrane (cyan) and BiFC stained cells. Green fluorescence indicates interaction-dependent regeneration of the Venus protein. Constructs are shown below. Here, the hexagons symbolize the native N-termini including mitochondrial localization sequences.</p

A model of RsfA action.

<p>In rich medium and during exponential growth, RsfA is either not present or not active, so that protein synthesis is fully active. In starving cells, RsfA binds to ribosomal L14 and, as a consequence, blocks ribosomal subunit joining and thus protein synthesis.</p

Mapping the RsfA binding site on ribosomal protein L14.

<p>(A) L14 in the context of the 3D structure of the 50S ribosomal subunit (a) (PDB: 2AWB) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815-Schuwirth1" target="_blank">[14]</a>. (b) Conserved residues of L14: magenta (highly conserved), grey (moderately conserved), turquoise (little or no conservation). (c) Mutated residues for interaction epitope mapping (red or green); residues involved in (red colors) and not involved (green colors) in RsfA-binding based on results from subfigure (C). (d) Residues of L14 highlighted that are involved in formation of intersubunit bridges with the 16S rRNA of the 30S subunit (bridge B5 (green colors), bridge B8 (red colors)) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815-Gao1" target="_blank">[15]</a>. (B) A docking model of L14 on the <i>E. coli</i> 50S subunit with bound RsfA. Critical L14 residues that mediate RsfA interaction (or that contact 16S rRNA) are colored in red according to A(c) and A(d). When RsfA is bound to L14 on a 50S subunit, 30S subunit joining is sterically blocked, clearly visible in B(b) as shown by the structural overlap of RsfA (dark blue) and the 30S subunit. A model of the ribosome with bound RsfA is available as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815.s001" target="_blank">Dataset S1</a>. (C) L14 interaction epitope mapping. Amino acids (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen-1002815-g002" target="_blank">Figure 2A(c)</a>) were mutated to alanine and the constructs tested by Y2H experiments. WT, wild type L14 construct; mutated residues and their positions are indicated. In the experiment, all bait constructs were simultaneously tested for reporter gene self-activation. No construct resulted in self-activation (data not shown). T97A, R98A, or K114A mutations (highlighted by arrows) abolished or weakened RsfA binding as indicated by 3-AT titrations; all other tested L14 mutation constructs are comparable to wild type L14.</p

RsfA inhibits translation by blocking ribosomal subunit joining.

<p>(A) Oligo(Phe) synthesis in a pure system containing pre-charged Phe-tRNAs (ten times over ribosomes), 30S and 50S subunits and the purified factors EF-Tu, EF-Ts and EF-G plus/minus RsfA from <i>E. coli</i>, 100% corresponds to 7 Phe incorporated per ribosome. Left panel, when indicated RsfA was added to the 50S subunits, before 30S subunits were added starting oligo(Phe) synthesis. Right panel, AcPhe-tRNA was bound to 70S ribosomes in the presence of poly(U) before the addition of RsfA. (B) Sister-aliquots from the same samples shown in (A) were analyzed on a sucrose gradient before oligo(Phe) synthesis. The presence of RsfA significantly reduces the fraction of 70S ribosomes. (C) Oligo(Phe)-synthesis as in (A) but with purified mitochondrial components (pig liver) and human mtRsfA (C7orf30). 39S and 28S indicate the large and small ribosomal subunits, 55S the associated mitochondrial ribosomes. For details see Experimental Procedures.</p