Human PAPS Synthase Isoforms Are Dynamically Regulated Enzymes with Access to Nucleus and Cytoplasm

In higher eukaryotes, PAPS synthases are the only enzymes producing the essential sulphate-donor 3′-phospho-adenosine-5′-phosphosulphate (PAPS). Recently, PAPS synthases have been associated with several genetic diseases and retroviral infection. To improve our understanding of their pathobiological functions, we analysed the intracellular localisation of the two human PAPS synthases, PAPSS1 and PAPSS2. For both enzymes, we observed pronounced heterogeneity in their subcellular localisation. PAPSS1 was predominantly nuclear, whereas PAPSS2 localised mainly within the cytoplasm. Treatment with the nuclear export inhibitor leptomycin B had little effect on their localisation. However, a mutagenesis screen revealed an Arg-Arg motif at the kinase interface exhibiting export activity. Notably, both isoforms contain a conserved N-terminal basic Lys-Lys-Xaa-Lys motif indispensable for their nuclear localisation. This nuclear localisation signal was more efficient in PAPSS1 than in PAPSS2. The activities of the identified localisation signals were confirmed by microinjection studies. Collectively, we describe unusual localisation signals of both PAPS synthase isoforms, mobile enzymes capable of executing their function in the cytoplasm as well as in the nucleus

C(P)XCG Proteins of <i>Haloferax volcanii</i> with Predicted Zinc Finger Domains: The Majority Bind Zinc, but Several Do Not

In recent years, interest in very small proteins (µ-proteins) has increased significantly, and they were found to fulfill important functions in all prokaryotic and eukaryotic species. The halophilic archaeon Haloferax volcanii encodes about 400 µ-proteins of less than 70 amino acids, 49 of which contain at least two C(P)XCG motifs and are, thus, predicted zinc finger proteins. The determination of the NMR solution structure of HVO_2753 revealed that only one of two predicted zinc fingers actually bound zinc, while a second one was metal-free. Therefore, the aim of the current study was the homologous production of additional C(P)XCG proteins and the quantification of their zinc content. Attempts to produce 31 proteins failed, underscoring the particular difficulties of working with µ-proteins. In total, 14 proteins could be produced and purified, and the zinc content was determined. Only nine proteins complexed zinc, while five proteins were zinc-free. Three of the latter could be analyzed using ESI-MS and were found to contain another metal, most likely cobalt or nickel. Therefore, at least in haloarchaea, the variability of predicted C(P)XCG zinc finger motifs is higher than anticipated, and they can be metal-free, bind zinc, or bind another metal. Notably, AlphaFold2 cannot correctly predict whether or not the four cysteines have the tetrahedral configuration that is a prerequisite for metal binding

Noninvasive characterization methods for ultra-short laser pulse induced volume modifications

We present two noninvasive characterization methods to investigate laser induced modifications in bulk fused silica glasses. The methods discussed are immersion microscopy and scanning acoustic microscopy (SAM). SAM shows merits in measuring the distance from sample surface to the first detectable density change of the modification, while immersion microscopy offers a look into the modification. Both noninvasive methods are preferred over conventional polishing or etching techniques due to the facts, that multiple investigations can be done with only one sample and lower time expenditure. The type II modifications were introduced by focusing laser pulses with high repetition rates into the fused silica

Measurement of the bound-electron g-factor difference in coupled ions

Quantum electrodynamics (QED) is one of the most fundamental theories of physics and has been shown to be in excellent agreement with experimental results(1–5). In particular, measurements of the electron’s magnetic moment (or g factor) of highly charged ions in Penning traps provide a stringent probe for QED, which allows testing of the standard model in the strongest electromagnetic fields(6). When studying the differences between isotopes, many common QED contributions cancel owing to the identical electron configuration, making it possible to resolve the intricate effects stemming from the nuclear differences. Experimentally, however, this quickly becomes limited, particularly by the precision of the ion masses or the magnetic field stability(7). Here we report on a measurement technique that overcomes these limitations by co-trapping two highly charged ions and measuring the difference in their g factors directly. We apply a dual Ramsey-type measurement scheme with the ions locked on a common magnetron orbit(8), separated by only a few hundred micrometres, to coherently extract the spin precession frequency difference. We have measured the isotopic shift of the bound-electron g factor of the isotopes (20)Ne(9+) and (22)Ne(9+) to 0.56-parts-per-trillion (5.6 × 10(−13)) precision relative to their g factors, an improvement of about two orders of magnitude compared with state-of-the-art techniques(7). This resolves the QED contribution to the nuclear recoil, accurately validates the corresponding theory and offers an alternative approach to set constraints on new physics

Supporting evidence for an alternative mouse PAPSS2 N-terminus.

<p>The mouse EST database at GenBank was searched using BLASTN using the following exon2 sequence of murine PAPS synthase: <i>gaccagcaaa aatccaccaa tgtggtctac caggcccatc atgtgagcag gaacaagaga ggacaagtgg ttggaaccag gggaggattc cgaggatgta ccgtgtggct aaca</i>.</p><p>All 15 hits are listed in this table. Moreover, the available cDNAs are listed. EST, expressed sequence tag.</p

Localisation of PAPS synthase wild-type fusion proteins.

<p><b>A and B</b>, microscopic images of PAPSS1- and PAPSS2-EGFP fusions. Transfected HeLa cells were fixed upon 24 h of fluorescent protein expression. Within every sample, cells with varying protein localisation could be observed. <b>C</b>, schematic for classifying localisation of PAPS synthase fusion proteins and their mutants. <b>D</b>, localisation pattern for PAPS synthase wild-type proteins with respect to the way of fixation and the expression time. A total of 1026 cells for PAPSS1-EGFP (P1) and 1942 cells for PAPSS2B-EGFP (P2) were evaluated, respectively, in fixed HeLa cells after 24 hours of expression. For all other conditions at least 200 cells were scored except PAPSS2-EGFP in living HeLa cells (n = 122). EGFP, enhanced green fluorescent protein.</p

A positively charged helical structure in PAPSS1 shows export activity.

<p><b>A</b>, alignment of residues Ser102-Gly124 of PAPSS1 (P1) and the respective Ser92-Gly114 sequence of PAPSS2 (P2). The two arginine residues targeted by alanine mutation are highlighted. NPS secondary structure consensus prediction confirms an extended α-helical conformation; “.”, coil; h/H, weak/prominent helical propensity. <b>B</b>, mapping of this motif (cyan) on the interface of the APS kinase dimer within the PAPSS1 crystal structure 1X6V. The two arginine residues are shown in stick representation (blue). Additionally, Glu108 is shown in stick representation (cyan). This residue may be necessary for charge compensation. <b>C</b>, a recombinant GST-PAPSS1-Helix-GFP protein showed export activity upon microinjection into the nuclei of Vero cells at the indicated time points (left panels). In contrast, mutation of two arginine residues completely abolished nuclear export (middle panel), and the respective substrate remained nuclear as microinjected GST-GFP alone under the same experimental conditions (right panel). As a positive control, the Rev NES of HIV (LQLPPLERLTL) was completely exported from the nucleus within 30 min following microinjection. Approximately 50 cells were injected, and representative examples are shown.</p

Apparent molecular weight of PAPSS1 mutants.

<p>All proteins were loaded at a concentration of 2–10 µM on the gel filtration column. Dilution during chromatography is three-fold at most. Co-localisation was assessed after binarisation of images as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029559#s3" target="_blank">methods</a> section.</p

A conserved NLS within the N-terminus of PAPS synthases.

<p><b>A</b>, alignment of the N-terminal protein sequence of PAPS synthases from several organisms. PAPS synthase N-termini were aligned with regard to their conserved N-terminal KKxK motif and the beginning of the conserved sequence of the globular APS kinase domain. Although this motif should be regarded as K(K/R)x(K/R) only due to the sequences from fruitfly and worm, we adhere to KKxK in the text for simplicity. All positively charged amino acids are written in red and underlined. An exon-exon border is indicated for all sequences derived from Ensembl. For mouse PAPS synthase 2 an alternative N-terminal sequence, P2 mouse*, is given that is supported by 14 different EST sequences. The sequence for the swapping construct of the PAPSS1 N-terminal sequence and the remaining part (body) of PAPSS2 is given below. Sequences used: human PAPSS1 (P1) [RefSeq: NM_005443], mouse P1 [RefSeq: NM_011863], rat P1 [RefSeq: NM_001106471], medaka P1 [Ensembl: ENSORLP00000008587], splice form RA from <i>Drosophila melanogaster</i> [FlyBase: FBgn0020389], the PPS-1 protein from <i>C. elegans</i> [RefSeq: NM_069456/Wormbase: T14G10.1], medaka P2 [Ensembl: ENSORLG00000006251], rat P2 [RefSeq: NM_001106375], mouse P2 [RefSeq: NM_011864<b>.3</b>], mouse P2* (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029559#pone.0029559.s001" target="_blank">Information S1</a></b>) and human PAPSS2B [RefSeq: NM_001015880]. <b>B</b>, HeLa cells expressing PAPS synthase point mutants within the conserved KKxK motif as EGFP fusions. All samples were fixed after 24 h expression. <b>C</b>, quantitative evaluation of mutants shown in B and comparison to the wild-type proteins PAPSS1-EGFP and PAPSS2-EGFP. <b>D and E</b>, a swapping construct with the N-terminal sequence of PAPSS1 and the PAPSS2-EGFP body expressed in HeLa cells shows clear nuclear accumulation relative to the PAPSS2-EGFP wild type.</p