Condensin subunits co-purify with MAP::SMCL-1.

<p>(A-B) Proteins that co-purified with MAP::SMCL-1 but not untagged control adult extracts, identified by tandem affinity purification and MudPIT mass spectrometry. Numbers represent average NSAF values from two replicas. Co-purified proteins with the highest NSAF values are shown in (A), values for other condensin subunits are shown in (B), and all other proteins are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#pgen.1006614.s011" target="_blank">S5 Table</a>. Condensin SMC subunits are highlighted.</p

SMCL-1 expression and protein features.

<p>(A) Adult hermaphrodites from wild-type (WT) and a strain carrying the <i>map</i>::<i>smcl-1</i> transgene driven by endogenous <i>smcl-1</i> 5’ and 3’ elements. A section of the germline is shown, imaged by DIC to show structures and fluorescent microscopy to detect mVenus expression from the MAP tag. Arrowheads denote the first four oocytes. (B) A typical SMC protein folds back on itself at a hinge domain, bringing coil regions together and creating a “head domain” (yellow) from ATPase domains in the N- and C-termini. SMCL-1 lacks predicted coil and hinge domains, but has N- and C-terminal ATPase domains that may be capable of forming a head domain (purple). (C) SMC head domain and the ATPase cycle, showing binding of ATP (red circle), ATP-dependent engagement of heads from two SMC proteins, and disengagement upon ATP hydrolysis. (D) SMCL-1 amino acid sequence aligned to <i>C</i>. <i>elegans</i> condensin SMC proteins. Shown are regions surrounding three conserved motifs found in SMCs and related ATPases: the Walker A motif, ABC transporter signature motif, and Walker B motifs, and their consensus sequences. SMCL-1 shares a conserved Walker A motif, but differs from consensus signature motif and Walker B motif at residues shown in red. Asterisk denotes catalytic amino acid required for ATP hydrolysis. x = any amino acid and h = hydrophobic amino acid.</p

SMCL-1 overexpression in the gut disrupts condensin I^DC localization on the X chromosomes.

<p>(A) Heat shock regimen for data in (B-E). Bolt represents the single heat-shock pulse given to young adult hermaphrodites from the wild-type or inducible <i>hs</i>:<i>smcl-1(+)</i> transgenic strain. (B-E) Adult hermaphrodite gut tissue of the indicated strain and treatment was stained with DAPI to image DNA (green in merge) and immuno-stained with antibody against CAPG-1(B-C), DPY-27 (D) and DPY-28 (E), (red in merges). Antibody against SMCL-1 was also included in (B and C), showing overexpression upon heat shock (blue in merge). The foci of staining created by condensin I^DC subunit association with the X chromosomes are lost when SMCL-1 is overexpressed. (C) A mosaic animal in which anti-SMCL-1 staining indicates that one cell lacks the transgene (left) and shows foci of anti-CAPG-1, while a neighboring cells has the <i>smcl-1</i> overexpression transgene (right) and CAPG-1 staining is weak and not localized to foci (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#pgen.1006614.s006" target="_blank">S6C and S6D Fig</a>). HS = heat shock.</p

Presence of predicted orthologs of SMCL-1, DPY-27 (I^DC), and SMC-4 (I & II) in various species.

<p>Phylogenetic tree built from all available <i>Caenorhabditis</i> species with sequenced and well-assembled genomes, other selected nematode species, and other selected model organisms. “<i>+”</i> symbol denotes the presence of SMCL-1-like protein based on similarity in a BLAST search and the additional criteria of short length, imperfect signature motif, and a Walker B motif lacking the catalytic glutamate (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#sec014" target="_blank">Methods</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#pgen.1006614.s003" target="_blank">S3 Fig</a>). “1” denotes orthologs detected using a high-confidence Ensemble-COMPARA method and “2” denotes orthologs detected using BLAST-neighbor-joining tree methods.</p

Heat-Shock Response Transcriptional Program Enables High-Yield and High-Quality Recombinant Protein Production in <i>Escherichia coli</i>

The biosynthesis of soluble, properly folded recombinant proteins in large quantities from <i>Escherichia coli</i> is desirable for academic research and industrial protein production. The basal <i>E. coli</i> protein homeostasis (proteostasis) network capacity is often insufficient to efficiently fold overexpressed proteins. Herein we demonstrate that a transcriptionally reprogrammed <i>E. coli</i> proteostasis network is generally superior for producing soluble, folded, and functional recombinant proteins. Reprogramming is accomplished by overexpressing a negative feedback deficient heat-shock response transcription factor before and during overexpression of the protein-of-interest. The advantage of transcriptional reprogramming versus simply overexpressing select proteostasis network components (e.g., chaperones and co-chaperones, which has been explored previously) is that a large number of proteostasis network components are upregulated at their evolved stoichiometry, thus maintaining the system capabilities of the proteostasis network that are currently incompletely understood. Transcriptional proteostasis network reprogramming mediated by stress-responsive signaling in the absence of stress should also be useful for protein production in other cells

Single-Step Inline Hydroxyapatite Enrichment Facilitates Identification and Quantitation of Phosphopeptides from Mass-Limited Proteomes with MudPIT

Herein we report the characterization and optimization of single-step inline enrichment of phosphopeptides directly from small amounts of whole cell and tissue lysates (100–500 μg) using a hydroxyapatite (HAP) microcolumn and Multidimensional Protein Identification Technology (MudPIT). In comparison to a triplicate HILIC-IMAC phosphopeptide enrichment study, ∼80% of the phosphopeptides identified using HAP-MudPIT were unique. Similarly, analysis of the consensus phosphorylation motifs between the two enrichment methods illustrates the complementarity of calcium- and iron-based enrichment methods and the higher sensitivity and selectivity of HAP-MudPIT for acidic motifs. We demonstrate how the identification of more multiply phosphorylated peptides from HAP-MudPIT can be used to quantify phosphorylation cooperativity. Through optimization of HAP-MudPIT on a whole cell lysate we routinely achieved identification and quantification of ca. 1000 phosphopeptides from a ∼1 h enrichment and 12 h MudPIT analysis on small quantities of material. Finally, we applied this optimized method to identify phosphorylation sites from a mass-limited mouse brain region, the amygdala (200–500 μg), identifying up to 4000 phosphopeptides per run

Single-Step Inline Hydroxyapatite Enrichment Facilitates Identification and Quantitation of Phosphopeptides from Mass-Limited Proteomes with MudPIT

Herein we report the characterization and optimization of single-step inline enrichment of phosphopeptides directly from small amounts of whole cell and tissue lysates (100–500 μg) using a hydroxyapatite (HAP) microcolumn and Multidimensional Protein Identification Technology (MudPIT). In comparison to a triplicate HILIC-IMAC phosphopeptide enrichment study, ∼80% of the phosphopeptides identified using HAP-MudPIT were unique. Similarly, analysis of the consensus phosphorylation motifs between the two enrichment methods illustrates the complementarity of calcium- and iron-based enrichment methods and the higher sensitivity and selectivity of HAP-MudPIT for acidic motifs. We demonstrate how the identification of more multiply phosphorylated peptides from HAP-MudPIT can be used to quantify phosphorylation cooperativity. Through optimization of HAP-MudPIT on a whole cell lysate we routinely achieved identification and quantification of ca. 1000 phosphopeptides from a ∼1 h enrichment and 12 h MudPIT analysis on small quantities of material. Finally, we applied this optimized method to identify phosphorylation sites from a mass-limited mouse brain region, the amygdala (200–500 μg), identifying up to 4000 phosphopeptides per run