Kinetic parameters of the N-acetyltransferase-His8Flag.

1<p>Concentrations were varied between 0.01 and 0.5 mM while the AcCoA concentration was fixed at 2 mM.</p>2<p>Concentrations were varied between 0.083 and 2 mM while the MU-GlcNH₂ concentration was fixed at 1 mM.</p

Immobilized [3H]acetylated N-acetyltransferase-His8Flag intermediate cannot be detected in immunoprecipitation experiments with anti-Flag M2 beads.

1<p>Anti-Flag beads were incubated with extracts from HeLa cell expressing (Transfected) or not expressing (Untransfected) N-acetyltransferase-His8Flag, washed and then incubated with [3H]acetyl-CoA for the given time and pH, at room temperature and with or without 1.3 mM of lipids containing 20% PI.</p>2<p>N-acetyltransferase activity in nmoles of MU produced/hour, and the concentration of protein (pmoles), calculated based on the specific activity of the purified transferase, are given.</p>3<p>The data represent three independent beads binding experiments and assays.</p>4<p>Not detectable.</p

Western blot analysis of the endogenous N-acetyltransferase protein in three human fibroblast cell lines.

<p>Fibroblast from an unaffected individual (WT), a patient with I-Cell disease, and a MPS IIIC patient were extracted with 1% DDM, 20 µg each of extracted protein were separated by SDS-PAGE, and the N-acetyltransferase proteins visualized using the N-terminal antibody. The blot was reprobed with anti GAPDH as a loading control. The specific activity of each extract is shown at the bottom, ND is not detected.</p

Effects of lipids on N-acetyltransferase activity are illustrated.

<p>Each set of data points represents the average of triplicate determinations of N-acetyltransferase activity with their standard deviations (SD) shown as error bars (A) DDM extracts from permanently transfected HeLa cells were serially diluted in CP buffer, pH 5.5 (filled diamonds), CP buffer containing 0.25% HSA (filled squares) or CP buffer containing 1.3 mM 20% PI (filled circles with the error bars representing SD too small to see, connected by the best fit line, R = 1) (X-axis, total extracted protein; Y-axis, transferase activity in nmol/h). (B) Equal amounts of anti-Flag-column purified N-acetyltransferase were assayed in the presence of increasing concentrations of lipids (X-axis, mM) with decreasing mole% of negatively charged PI (40-0% PI+40–80% PC+20% CH); 40% PI, darkly shaded bars; 20% PI, grey hatched bars; or PC (0% PI), white bars. (C) Stability of identical amounts of purified N-acetyltransferase in CP buffer pH 5.5, containing either 0.25% (w/v) HSA (white bars) or 1.3 mM, 20% PI, lipids (shaded bars) (X-axis, hours at 37°C; Y-axis transferase activity in nmol/h).</p

The protein expressed by a construct encoding myc-N-acetyltransferase-His8Flag was analyzed by Western blotting.

<p>(A) Extracts from HeLa cells permanently expressing the singly-tagged N-acetyltransferase-His8Flag (lane 1) were compared with HeLa cell transiently expressing myc-N-acetyltransferase-His8Flag (lane 2) using the N-terminal N-acetyltransferase antibody. The levels of transferase activity in the extracts are given below each lane. (B) The oligosaccharides present on the doubly-tagged myc-N-acetyltransferase-His8Flag protein (lane 1) were analyzed based on endo-H (lane 2) and/or PNGase (lane 3) sensitivities by Western blotting using a myc antibody.</p

Western blot analysis of the post nuclear supernatant (PNS) and enriched lysosomal (Lys) fractions separated magnetically from extracts of N-acetyltransferase-His8Flag permanently transfected HeLa cells loaded with iron-dextran (FeDex).

<p>After SDS-PAGE the proteins in the PNS and Lys fractions (1 µg total protein from each fraction was loaded) were visualized with the N-terminal N-acetyltransferase antibody (N-term). The nonspecific ∼50 kDa band visualized with the N-terminal antibody is marked as “X”. As control for the enrichment of lysosomes in the “Lys” fraction, the blot was stripped and re-probed with an antibody against human lysosomal ß-hexosaminidase A (Hex A). Furthermore, markers for the ER (Calnexin) and early endosome (EEA1) were visualized after stripping and re-probing the blot with the appropriate antibody (bottom).</p

Scaled diagrams of the full length N-acetyltransferase and the small N-terminal loop fragment of the protein expressed by the cDNA constructs used in this study.

<p>Each construct also encodes a C-terminal His8-Flag tag. Predicted masses (kDa) are shown for each protein segment. TMDs are indicated by hatched rectangles, soluble loops that are predicted to reside in the ER/lysosomal lumen are shown as clear boxes and those predicted to reside in the cytosol as shaded boxes. Putative Asn-linked glycosylation sites (NXS/T) are shown as “*”. The location and ends of the peptide sequence used to generate the “N-terminal” antibody and utilized in this report, are shown between the two panels. (A) Full length N-acetyltransferase with both the long signal peptide (SP-L), found in the genomic sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024951#pone.0024951-Durand1" target="_blank">[13]</a>, and the short signal peptide (SP-S), used in this report and found in EST data bases are indicated. The positions and sequences of previously published intracellular transport signals <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024951#pone.0024951-Durand1" target="_blank">[13]</a> are shown, as are the N-termini, identified in this report, of the processed alpha- and ß- chains. (B) The N-terminal fragment containing the short signal peptide and the first luminal loop of N-acetyltransferase are depicted.</p

Proteins extracted from HeLa cells permanently expressing N-acetyltransferase-His8Flag were separated by molecular size exclusion chromatography on a 90×1.5 cm Sephacryl S-400 column eluted at 4°C.

<p>Each 1 mL fraction (X-axis) was analyzed for transferase activity (nmol/mL* h, left Y-axis, solid line) and total protein (µg/mL, right Y-axis, dashed line). Additionally every fifth fraction was analyzed for N-acetyltransferase protein by dot-blot using the N-terminal antibody (shown below the X-axis).</p

Direct Michaelis-Menten best-fit curves of specific activity (nmol/h*ng) versus [S] for the N-acetyltransferase reaction with graphic insets displaying the corresponding Lineweaver-Burk double reciprocal plots.

<p>Anti-flag affinity column purified N-acetyltransferase was used and each datum point represents the average of two or three assays. (A) The concentration of MU-GlcNH₂ was varied between 0.01 and 1 mM, while AcCoA was fixed at 0.17 mM (▴), 0.33 mM(○) or 1.0 mM (▪). (B) The concentration of AcCoA was varied between 0.083 and 2.0 mM while MU-GlcNH₂ was fixed at 0.05 mM (⧫), 0.10 mM (□) or 0.30 mM (•). The experiment was independently repeated three times with a representative set of graphics shown.</p

The effects of detergent extraction procedures on the intensity of bands associated with monomeric N-acetyltransferase (i.e., 62 and 44 kDa), detected by Western blotting with the C-terminal rabbit Flag antibody.

<p>HeLa cells, permanently transfected with N-acetyltransferase-His8Flag, were; lane 1- first extracted with 1% DDM, centrifuged and the extract denatured in SDS-PAGE sample buffer (containing a reducing agent) at room temperature; lane 2- homogenized (in water), sonicated and boiled in lithium dodecyl sulfate reducing sample buffer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024951#pone.0024951-Durand1" target="_blank">[13]</a>; or lane 3- extracted with 2% SDS, diluted with SDS-PAGE reducing sample buffer and heated at 65°C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024951#pone.0024951-Fedele1" target="_blank">[6]</a>. Bottom panel shows the loading control, the cation-independent mannose-6-phosphate receptor, which contains a single TMD.</p