Information S1 - Human PAPS Synthase Isoforms Are Dynamically Regulated Enzymes with Access to Nucleus and Cytoplasm

Cellular localisation of PAPS synthase proteins. Localisation of different PAPS synthase fusion proteins in several cell types. The influence of the nature of the fusion protein (expressed in HeLa cells) as well as the cell line used was assayed for PAPSS1 and PAPSS2 fusion proteins. At least 200 cells were scored according to the schematic presented in Figure 1. PAPS synthases contain a leucine-rich motif that acts as weak NES. A, alignment of residues Leu254–Leu270 of PAPSS1 (P1) with the respective Leu244–Leu260 sequence of PAPSS2 (P2). The short and long peptidic motifs tested for their nuclear export activity are indicated. Residues of a putative leucine-rich NES are written in bold. NPS secondary structure consensus prediction is shown below the alignment; “.”, coil; h/H, weak/prominent helical propensity; “?”, no prediction. B, mapping of this putative leucine-rich export signal on one part of the ATP sulphurylase domain of the PAPSS1 crystal structure 1X6V. The short sequence is surface-exposed and shown in cyan, the extension in blue. C, recombinant GST-PAPSS1/2-NES-GFP proteins containing the respective leucine-rich signal sequences showed weak export activity upon microinjection into the nuclei of Vero cells at the indicated time points (left panels). In contrast, the GST-GFP substrate remained nuclear under the same experimental conditions (right panel). Approximately 50 cells were injected, and representative images from live-cell fluorescence microscopy are shown. D, due to the slow export kinetics of this motif it was not possible to monitor completion of export within one round of the cell cycle, therefore monitoring included mitotic nuclear envelope breakdown and reassembly. Expression levels of PAPSS2-EGFP wild-type and mutant proteins. Equal amounts of HeLa cells were transfected with wild-type PAPSS2-EGFP expression plasmid as well as those three mutants that showed the largest shifts in cellular localisation: KK6,8AA, RR101,102AA and LL252,254AA and expression levels were assessed by western blotting using an anti-GFP antibody. Equal loading was shown using an anti-tubulin antibody. All four fusion proteins showed similar expression levels at comparable rates of transfection. Additionally, detailed information on the plasmids and oligonucleotides used in this study can be found within Information S1.

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