Primers used for site directed mutagenesis.

<p>Primers used for site directed mutagenesis.</p

Analysis of the interacting properties of different domains of R6 by immunoprecipitation (GFP-Trap) in mammalian cells.

<p>Hek293 cells were transiently transfected with expression vectors coding for YFP, YFP-R6 wild type, and the corresponding mutants YFP-R6 RARA and YFP-R6 RAHA (A), YFP-R6 WDNAD and YFP-R6 WANNA (B), or YFP-R6 S25A and YFP-R6 S74A plasmids (C). Immunoprecipitation analyses were performed using GFP-Trap system (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131476#sec002" target="_blank">Materials and Methods</a> section). 40 μL of eluted beads and thirty micrograms of total protein from the soluble fraction of cell lysates (input) were analyzed by SDS-PAGE and Western blotting using appropriated antibodies.</p

Glycogenic activity of different mutated forms of R6.

<p>(A) Measurement of glycogenic activity of different R6 mutated forms. N2a cells were transfected using 1 μg of pFLAG plasmid (negative control), pFLAG-R6 plasmid or its corresponding mutants. Forty-eight hours after transfection, the amount of glycogen was determined as described in Materials and Methods and represented as μg of glucose/mg of protein/relative amount of R6 respect to actin (wild type value considered as 1). Bars indicate standard deviation of three independent experiments (**p<0.01 or ***p<0.001, compared with control cells transfected with an empty plasmid; ##p<0.01, compared with cells expressing R6-WT). An inset with the mean values +/- standard deviation is included. (B) Protein levels of FLAG-R6 forms. A representative western blot analysis is shown. Cell extracts (30 μg) were analyzed using the corresponding anti-FLAG and anti-actin antibodies.</p

Binding of 14-3-3 proteins to R6 prevents its lysosomal degradation.

<p>A) R6-S74A mutant possesses a shorter half-life than wild type protein. Hek293 cells were transfected with pFLAG-R6 wt or pFLAG-R6-S74A plasmids. 24 hours after transfection, cells were treated with cycloheximide (300 μM) to block protein synthesis. At the indicated times, cell extracts (30 μg) were analyzed by Western blotting using anti-FLAG and anti-tubulin (as loading control). A representative western blot is shown on the left panel. On the right panel, the intensity of the bands related to the levels of tubulin is plotted and normalized respect to the values at time 0 (bars indicate the standard deviation of at least three independent experiments; **p < 0.01). B) R6-S74A protein is degraded by the lysosomal pathway. Hek293 cells were transfected with pFLAG-R6-S74A plasmid. Eighteen hours after transfection, cells were treated with ammonium chloride (20 mM)/ leupeptin (100 μM) or MG132 (5 μM) for six hours. Then, cells were lysed and extracts (30 μg) were analyzed by immunoblotting using anti-FLAG antibody and anti-tubulin as loading control. The intensity of the bands related to the levels of tubulin is plotted (bars indicate standard deviation of at least three independent experiments; *p < 0.05).</p

Subcellular localization of R6 S25A and R6 S74A mutated forms.

<p>N2a cells were transfected with pYFP empty plasmid, pYFP-R6 wild type, pYFP-R6 S25A or pYFP-R6 S74A plasmids. The subcellular localization of R6 forms and glycogen granules was carried out as described in Materials and Methods. Images were obtained by using confocal microscopy (bars indicate 10 μm). Images corresponding to visible, YFP (in yellow) and glycogen (in red) fluorescences are shown.</p

Schematic representation of the different binding regions in R6.

<p>R6 possesses three separated interaction domains: PP1c binding motif (R₁₀₂VRF), PP1 substrate binding region (W₂₆₇DNND) and 14-3-3 binding motif (RARS₇₄LP). GS, glycogen synthase; GP, glycogen phosphorylase.</p

Analysis of the interacting properties of different domains of R6 by yeast two-hybrid analyses.

<p>Upper panels: yeast THY-AP4 strain was transformed with plasmids pBTM-R6 wt (LexA-R6), pBTM-R6 RARA and pBTM-R6 RAHA (A), pBTM-R6 WDNAD, and pBTM-WANNA (B) or pBTM-R6 S25A and pBTM-R6 S74A (C) and with pACT2 (GAD), pACT2-PP1α (GAD-PP1α), pACT2-laforin (GAD-laforin) or pACT2-14-3-3ε (GAD-14-3-3ε). Protein interaction was estimated by measuring the β-galactosidase activity. Values correspond to means from at least 6 different transformants (bars indicate standard deviation). Lower panels: protein expression in yeast transformants was analyzed by Western blotting using anti-HA antibodies (for the GAD-fusions) and anti-LexA (for the LexA-fusions) in several transformants from each condition. A representative western blot of some of these transformants is shown.</p

Characterization and structural modelling of R6 domains.

<p>(A) Multiple sequence alignment of PPP1R3D (R6), PPP1R3C (R5) and PPP1R3B (GL) proteins sequences from <i>H</i>. <i>sapiens</i> (Uniprot entries: O95685, Q9UQK1 and Q86XI6, respectively) was performed using the Clustal Omega program (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131476#sec002" target="_blank">Materials and Methods</a>). Invariance and conservation are highlighted by black and gray shadowing respectively. Amino acid sequences for the binding to 14-3-3 proteins, PP1 catalytic subunit and PP1 glycogenic substrates are enclosed in yellow, orange and cyan boxes respectively. Brackets indicate the modeled sequence of R6 shown in panel B. The CBM21 domain of R6 (according to Uniprot) is underlined in orange. Red triangles point at the mutated residues obtained in this study. (B) Homology model of the CBM21 domain of R6 was based on the template of GL (pdb entry: 2EEF). The amino acids corresponding to the PP1 glycogenic substrate binding domain (W₂₆₇DNND) and to the putative RVXF (R₂₅₂VHF) are shown in sticks and colored in cyan or orange, respectively (left panel). The N- and C-terminus of the model is also indicated. A representation of the surface of the R6 homology model to show the exposed amino acids to the solvent is presented in the right panel. Images were generated using PyMol (DeLano Scientific LLC, USA).</p

Human laforin contains nine cysteines.

<p>(A) Multiple sequence alignment of laforin orthologs. Sequences of several vertebrate (indicated by the brackets) and two invertebrate (<i>N. vectensis</i> and <i>T. gondii</i>) organisms were used (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069523#pone.0069523.s001" target="_blank">Table S1</a> for details). The different cysteines are highlighted in yellow, and the position of cysteines in human laforin is marked (catalytic cysteine C266 appears in red). (B) Schematic of laforin domains (CBM: carbohydrate binding module; DSPD: dual-specificity phosphatase domain). The location of the nine cysteines is shown (blue numbers). (C) Tertiary structure prediction of the CBM (left) and DSPD (right) laforin domains. Homology models were created using the structures of glucoamylase (PDB: 1ACZ) and SEX4 (PDB: 3NME) as templates for the CBM and DSPD, respectively. The models were used to estimate the possible location of the cysteines in the tertiary structure. The cysteines studied in this work appear in yellow in the CBM and in orange in the DSPD; in grey, known tryptophans responsible of the carbohydrate binding.</p

Laforin-C329S and malin physically interact and form a functional complex in mammalian cells.

<p>(A) Yeast two-hybrid analysis. THY-AP4 yeast strain was transformed with pACT2-malin and LexA-laforin (WT or C329S) and the interaction was assessed by measuring the β-galactosidase activity. (B) Co-immunoprecipitation assay. HEK293 cells were co-transfected with plasmids myc-laforin (WT or C329S) and HA-malin. Cells were lysed and total lysates were incubated with anti-myc antibody and protein A/G beads. After washing, beads were boiled in loading buffer and purified proteins analyzed by SDS-PAGE and Western blot using anti-myc or anti-HA antibodies. (C) Ubiquitination analysis of R5/PTG by the laforin-malin complex. Overexpression of 6xHis-ubiquitin, pCMV-HA-malin, pCMV-myc-R5/PTG and pCMV-HA-laforin (wild type or C329S) in HEK293 cells, followed by lysis in presence of guanidinium chloride and purification of the ubiquitinated proteins by affinity chromatography using a cobalt resin. The result of the purification was analyzed using Western blot with anti-myc antibodies. Bound: proteins retained in the resin; crude extracts (50 µgr, C.E.) were immunodetected with anti-HA antibodies. *: polyubiquitinated forms.</p