Peptidase activity assay.

<p>(A) The peptidase activity of EcV with octapeptide activators. The blue and orange lines represent the activity of EcV in presence of EDLSRFIL and EDLSR<u>Y</u>IL peptides, respectively. The gray and yellow lines represent the activity of inactive ΔT1_EcV in presence of the same peptides. (B) Peptidase activity of the active form of EcV with EcU protein activators. The blue and orange lines represent the activity of EcV in the presence of wild-type EcU and F441Y mutant, respectively. The gray and yellow lines represent the activity of inactive ΔT1_EcV in presence of the same proteins. The brown and green lines represent the activity of EcV in the presence of P315T and P315T/F441Y double mutant, respectively. The error bars were calculated based on three independent experiments. The values are the means ± SD (n = 3).</p

Sequence comparisons among HslU proteins from three different biological kingdoms.

<p>(A) Sequence alignment of C-terminal segment and its neighboring region in HslUs from different organisms (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103027#s2" target="_blank">Materials and Methods</a> for detailed uniprot ID and species). The sequences are grouped in the order prokaryotic, archaeal, eukaryotic HslU1, and eukaryotic HslU2. Shading indicates residues that are identical (bold white in red-shaded box) or highly conserved (red in empty box) between species. Secondary structural elements are indicated above the sequence (α-helix, spring; β-strand, arrow). The mutated residues in this study are marked using a red star and the neighboring residues are marked using blue filled circles at the bottom. The sequence numbers for the aligned residues are also provided. (B) Phylogenetic tree of the aligned HslUs from panel (A). Prokaryotic HslUs are more similar to archael HslUs than to eukaryotic HslUs. There are two eukaryotic HslUs, of which HslU1 is relatively distant from the HslUs in the other kingdoms. The scale bar indicates amino acid substitutions per site.</p

Caseinolytic activity of EcVU complex.

<p>Active wild-type EcV as well as inactive ΔT1_EcV were used in control experiments. Wild-type and three mutant EcU proteins (F441Y, P315T, and P315T/F441Y) were used to activate EcV in the presence of ATP or ATPγS as indicated above. Note that the caseinolytic activity of EcVU complex with ATPγS is higher than that with ATP, however, the general trend is basically the same with the three mutants. The caseinolytic activity of wild-type EcVU complex at a fixed time point (12 h) was arbitrarily set at 100%. Each bar and line represents the mean and standard deviation values from three independently performed assays.</p

ATPase activity of EcU and its mutants in the presence or absence of EcV and ΔT1_EcV, respectively.

<p>The inactive ΔT1_EcV mutant was used in a control experiment as this variant is known to highly augment the ATPase activity of EcU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103027#pone.0103027-Park1" target="_blank">[23]</a>. Wild-type and three mutants (F441Y, P315T, and F441Y/P315T) were used for activating EcV. The ATPase activity of wild-type EcVU complex at a fixed time point (1 h) was arbitrarily set at 100%. Each bar and line represents the mean and standard deviation values from three independently performed assays.</p

MBP-SulA degradation activity.

<p>(A) Bands for MBP-SulA (substrate), HslU (wild-type EcU or EcU mutants), MBP (reaction product), and HslV (EcV or ΔT1_EcV) are indicated. All reactions were performed in the presence of ATP and SulA is degraded in the reaction mixtures containing active EcV. The control lane contained substrate MBP-SulA only. (B) The same experiments were performed as described in panel (A) in the absence of ATP, and did not yield the MBP product. (C) Time course analysis of the degradation of MBP-SulA by EcV and EcU in the presence of ATP. (D) As in (C), except that the EcU F441Y mutant was used. The F441Y mutant produced no enhancement of SulA degradation compared with wild-type. (E) SulA degradation activity of wild-type EcVU and EcV-EcU mutant complexes at a fixed time point (2 h).</p

Structure of the EcU and EcV interaction region.

<p>(A) Schema showing the superimposed apo-subunit of EcU (PDB ID: 1DO0), ATP-bound subunit of EcU (PDB ID: 1DO0), and EcV-bound EcU, which reveals the extended C-terminal tail. This EcVU complex structural model was generated using the HiVU structure as a template <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103027#pone.0103027-Sousa1" target="_blank">[18]</a>. In the apo EcU structure, one monomer of EcU is colored green, and its neighboring monomer is highlighted in sky blue for clarity. In the ATP-bound EcU structure, the two subunits are colored magenta and pink, respectively. In the schema for the HslVU complex, the two subunits are colored brown and yellow, respectively. The critical residues for the interaction between Phe441 and other amino acids are presented in a stick model. A prime annotation (′) is added for adjacent subunits of EcU. (B) The same view of the EcVU complex structure that includes the electrostatic potential of the EcV surface. The C-terminal tail of EcU extends toward a pocket formed by the two EcV subunits. The loop right before the Pro315 residue makes contact with EcV. The 3₁₀-helix starting with Pro315 is colored differently. Oxygen and nitrogen atoms are colored red and blue, respectively.</p

X-ray data collection and refinement.

<p>Values in parentheses are for the outer shell (2.41−2.33 Å) for ROCK(535–700), and (2.49−2.40 Å) for ROCK (535–709).</p>†<p><i>R</i>_sym  =  ∑_h∑_i| I_i(h) - | /∑_h∑_iI_i(h), where I_i(h) is the i^th measurement and is the mean of all measurements of I(h) for Miller indices h.</p>‡<p>R  =  ∑(| F_obs| - | F_calc|)/∑| F_obs|. <i>R</i>_free is obtained for a test set of reflections (5.3% of total).</p

Stereogram of electron density map at the coiled-coil interface in the region of the E613/R617/D620 bulge.

<p>Chain C is shown in yellow and chain D in grey. The σ_A weighted 2F_O – F_C map is contoured at 1.5 σ and computed anisotropically to 2.3 Å. Due to the anisotropy, the map was sharpened by applying a B factor scaling of -10 Ų (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018080#s4" target="_blank">methods</a>).</p

Overall structure of ROCK-CC.

<p>(A) Ribbon diagram of the ROCK-CC dimer is shown with chain A in green and chain B in cyan. The parallel coiled-coil homo-dimer is asymmetric. (B) Crystal packing of dimer A/B with dimer C/D. The orientation is that of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018080#pone-0018080-g003" target="_blank">Fig. 3A</a> turned 90° towards the viewer along the superhelical axis. Chain C is colored in yellow, chain D is in magenta.</p

Functional activity of yeast Ubr1 mimics of missense JBS-UBR1 mutants.

<p>(A) Relative enzymatic activity of βgal in extracts from <i>S. cerevisiae</i> JD55 (<i>ubr1Δ</i>) that expressed His-βgal or Tyr-βgal, and also carried an empty vector, or an otherwise identical plasmid expressing wild-type <i>S. cerevisiae</i> Ubr1, or (separately) its three missense mutants Ubr1^V146L, Ubr1^H160R, or Ubr1^Q1224E. The activity of βgal was measured in triplicates, with standard deviations shown. (B) Relative levels of induction of the peptide transporter Ptr2 were assayed by measuring the activity of a plasmid-borne <i>lacZ</i> (βgal-encoding) reporter that was expressed from the P<i>_PTR2</i> promoter in <i>ubr1Δ S. cerevisiae</i> that carried either an empty vector or otherwise identical plasmids that expressed either wild-type Ubr1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Xia1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Turner1" target="_blank">[52]</a> or its indicated mutants. Cells were grown to A₆₀₀ of ∼0.8 in SC(-Ura, -Leu) medium at 30°C, followed by measurements, in triplicate, of βgal activity in cell extracts, with standard deviations shown. (C) The lysine-requiring JD55 (<i>ubr1Δ</i>) <i>S. cerevisiae</i> strain was grown on plates containing 110 µM lysine (Lys) or 66 µM Lys-Ala dipeptide as the sole source of Lys in the medium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang3" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Turner1" target="_blank">[52]</a>. JD52 (<i>ubr1Δ</i>) cells carried a vector plasmid or otherwise identical plasmids expressing wild-type Ubr1 or its missense mutants Ubr1^H160R, Ubr1^V146L and Ubr1^Q1224E. Cells were grown to A₆₀₀ of ∼1 in SC(-Leu) medium at 30°C, washed in sterile water, serially diluted 5-fold, spotted on SC(-Leu, -Lys) plates containing 110 µM Lys or 66 µM Lys-Ala, and incubated at 30°C for 3 days. (D) Cell extracts (equal total protein levels) from experiments described in panels A and B were subjected to SDS-PAGE, followed by immunoblotting with affinity-purified anti-Ubr1 antibody (upper panel) and anti-tubulin antibody (a loading control; lower panel). Asterisk indicates a protein that crossreacts with anti-Ubr1 antibody. (E) Extracts from human lymphocytes (equal amounts of total protein) were subjected to SDS-PAGE, followed by immunoblotting with antibody to human UBR1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s4" target="_blank">Materials and Methods</a>). Lane 1, wild-type lymphocytes. Lane 2, same as lane 1 but from lymphocytes of patient #2 (see the main text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g002" target="_blank">2</a>). Lane 3, same as lane 1 but with lymphocytes from patient #3. Lane 4, same as lane 1, but with lymphocytes from a JBS patient with a homozygous nonsense mutation in <i>UBR1</i>, previously shown to have no detectable UBR1 (null UBR1 control) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Varshavsky3" target="_blank">[17]</a>. Lane 5, same as a lane 1.</p