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.