Structural Insight into Regulation of the Proteasome Ub-Receptor Rpn10

Ubiquitylation is a posttranslational modification that determines protein fate. The ubiquitin code is written by enzymatic cascades of E1 and E2 and E3 enzymes. Ubiquitylation can be edited or erased by deubiquitylating enzymes. Ub-receptors are proteins that read and decipher the ubiquitin codes into cellular response. They harbor a ubiquitin-binding domain and a response element. Interestingly, Ub-receptors are also regulated by ubiquitylation and deubiquitylation. However, until recently, the molecular details and the significance of this regulation remained enigmatic. Rpn10 is a Ub-receptor that shuttles ubiquitylated targets to the proteasome for degradation. Here we review recent data on Rpn10, with emphasis on its regulation by ubiquitylation

The Hetero-Hexameric Nature of a Chloroplast AAA+ FtsH Protease Contributes to Its Thermodynamic Stability

FtsH is an evolutionary conserved membrane-bound metalloprotease complex. While in most prokaryotes FtsH is encoded by a single gene, multiple FtsH genes are found in eukaryotes. Genetic and biochemical data suggest that the Arabidopsis chloroplast FtsH is a hetero-hexamer. This raises the question why photosynthetic organisms require a heteromeric complex, whereas in most bacteria a homomeric one is sufficient. To gain structural information of the possible complexes, the Arabidopsis FtsH2 (type B) and FtsH5 (type A) were modeled. An in silico study with mixed models of FtsH2/5 suggests that heteromeric hexamer structure with ratio of 4∶2 is more likely to exists. Specifically, calculation of the buried surface area at the interfaces between neighboring subunits revealed that a hetero-complex should be thermodynamically more stable than a homo-hexamer, due to the presence of additional hydrophobic and hydrophilic interactions. To biochemically assess this model, we generated Arabidopsis transgenic plants, expressing epitope-tagged FtsH2 and immuno-purified the protein. Mass-spectrometry analysis showed that FtsH2 is associated with FtsH1, FtsH5 and FtsH8. Interestingly, we found that ‘type B’ subunits (FtsH2 and FtsH8) were 2–3 fold more abundant than ‘type A’ (FtsH1 and FtsH5). The biochemical data corroborate the in silico model and suggest that the thylakoid FtsH hexamer is composed of two ‘type A’ and four ‘type B’ subunits

Chapter Structural Insight into Regulation of the Proteasome Ub-Receptor Rpn10

Ubiquitylation is a posttranslational modification that determines protein fate. The ubiquitin code is written by enzymatic cascades of E1 and E2 and E3 enzymes. Ubiquitylation can be edited or erased by deubiquitylating enzymes. Ub-receptors are proteins that read and decipher the ubiquitin codes into cellular response. They harbor a ubiquitin-binding domain and a response element. Interestingly, Ub-receptors are also regulated by ubiquitylation and deubiquitylation. However, until recently, the molecular details and the significance of this regulation remained enigmatic. Rpn10 is a Ub-receptor that shuttles ubiquitylated targets to the proteasome for degradation. Here we review recent data on Rpn10, with emphasis on its regulation by ubiquitylation

Chapter Structural Insight into Regulation of the Proteasome Ub-Receptor Rpn10

Ubiquitylation is a posttranslational modification that determines protein fate. The ubiquitin code is written by enzymatic cascades of E1 and E2 and E3 enzymes. Ubiquitylation can be edited or erased by deubiquitylating enzymes. Ub-receptors are proteins that read and decipher the ubiquitin codes into cellular response. They harbor a ubiquitin-binding domain and a response element. Interestingly, Ub-receptors are also regulated by ubiquitylation and deubiquitylation. However, until recently, the molecular details and the significance of this regulation remained enigmatic. Rpn10 is a Ub-receptor that shuttles ubiquitylated targets to the proteasome for degradation. Here we review recent data on Rpn10, with emphasis on its regulation by ubiquitylation

Models of the <i>Arabidopsis thaliana</i> FtsH complex.

<p>(<b>A</b>) TtFtsH (left), structure of <i>T. thermophilus</i> (PDB entry 2DHR) complex rendered as solvent accessible surface. Shown is the face of the protease side, with each chain in a different color. Models of the AtFtsH2 (middle) and AtFtsH2/5 rendered as TtFtsH. Chains of AtFtsH2 are colored in blue and chains of AtFtsH5 are colored in orange. (<b>B</b>) Summary of the calculated buried surface interfaces of the monomers in the <i>T. thermophilus</i>, AtFtsH2 and AtFtsH2/5 complexes. To facilitate the comparison, the values are color-coded as in <i>A</i>. (<b>C</b>) Zoom-in at the interface between chains <i>b</i> and <i>c</i>. The left and the right panels depict the homomeric (FtsH2) and the heteromeric (FtsH2/FtsH5) complexes, respectively. The picture in the left panel is centered at Arg-204 in chain <i>b</i> (marine blue), and its nearby residues in chain <i>c</i> (dark blue). Chain <i>c</i> of FtsH5 is colored in orange (like in 6A). Yellow dashed lines denote salt bridges; distances are indicated in Angstroms. Color code: oxygen – red; nitrogen – blue; carbon – yellow.</p

Immuno-purification of FtsH2-HA.

<p>Intact chloroplasts were first isolated from FtsH2-HA plants, then lysed and solubilized with 1% β-DM. Anti-HA agarose conjugate was added and washed extensively, before elution with 2× sample buffer. Samples from the different steps were resolved by SDS-PAGE, blotted and reacted with HA and FtsH antibodies.</p

Abundance and localization of the FtsH2-HA protein.

<p>(<b>A</b>) Immuno-blot analysis of total protein, extracted from transgenic and WT plants. Antibodies used are indicated on the left. (<b>B</b>) Intact chloroplasts were isolated from FtsH2-HA plants and fractionated into thylakoids and stroma. Samples were separated by SDS-PAGE, and gels were either stained by Coomassie blue or blotted onto membranes and reacted with anti-HA and anti-FtsH antibodies. All samples contained equivalents of 3 µg chlorophyll, except the HA blot, which contained equivalents of 10 µg chlorophyll. The antibodies used, or the location of specific proteins on the stained gels, are indicated on the left.</p

Characterization of transgenic plants expressing epitope-tagged FtsH2.

<p>(<b>A</b>) Schematic representation of the HA-tagged FtsH2 construct. A 3×HA tag sequence was cloned in-frame to the FtsH2 cDNA 3′ end. The construct was cloned between the 35S constitutive promoter and the OCS terminator. Arrowheads correspond to primers used in further analyses. (<b>B</b>) PCR on genomic DNA of kanamycin-resistant transgenic plants. Primers A and B (shown in <i>A</i>) were used to distinguish between WT and transgenic plants. (<b>C</b>) Qualitative RT-PCR on total RNA. Primers A and B were used to detect FtsH2-HA transcripts. Primers A and C were used to detect the native FtsH2 transcript. All other transgenic seedlings demonstrated a similar behavior. (<b>D</b>) Immuno-blot analysis of total protein from FtsH2-HA and WT plants. The blot was reacted with an HA antibody. Load is 10 µg chlorophyll per lane. All other transgenic seedlings demonstrated a similar behavior.</p

Mass spectrometry analysis of purified FtsH2-HA.

<p>Alignment of mature FtsH1, FtsH5, FtsH2 and FtsH8. Highlighted are peptides identified by MS. Yellow, peptides conserved between FtsH1 and FtsH5; green, peptides conserved between FtsH2 and FtsH8; blue, peptides specific to FtsH1; purple, peptides specific to FtsH5; olive, peptides specific to FtsH2; gray, peptides specific to FtsH8. Red Roman numerals indicate peptide groups used for quantification.</p

Relative abundance of FtsH subunits.

<p>The abundance of FtsH subunits was deduced from the peak area of specific peptides identified in the MS analysis. (<b>A</b>) Ratios between ‘type B’ and ‘type A’ subunits in isolated complexes (C) and intact thylakoids (T). The Roman numerals correspond to those indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036008#pone-0036008-g004" target="_blank">Figure 4</a>. The horizontal dashed lines represent possible ratios within the hexamer. (<b>B</b>) Ratios between the products of duplicated genes within a type. The sequences of the peptides used in the analysis are indicated above the bars. Values are averages ±s.d. of 2–4 replicates.</p