Analysis of Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL-4) mutant proteins reveals that S-acylation is required for the function of syntaxin 11 in natural killer cells

Natural killer (NK) cell secretory lysosome exocytosis and cytotoxicity are impaired in familial hemophagocytic lymphohistiocytosis type 4 (FHL-4), a disorder caused by mutations in the gene encoding the SNARE protein syntaxin 11. We show that syntaxin 11 binds to SNAP23 in NK cells and that this interaction is reduced by FHL-4 truncation and frameshift mutation proteins that delete all or part of the SNARE domain of syntaxin 11. In contrast the FHL-4 mutant proteins bound to the Sec-1/Munc18-like (SM) protein Munc18-2. We demonstrate that the C-terminal cysteine rich region of syntaxin 11, which is deleted in the FHL-4 mutants, is S-acylated. This posttranslational modification is required for the membrane association of syntaxin 11 and for its polarization to the immunological synapse in NK cells conjugated to target cells. Moreover, we show that Munc18-2 is recruited by syntaxin 11 to intracellular membranes in resting NK cells and to the immunological synapse in activated NK cells. This recruitment of Munc18-2 is abolished by deletion of the C-terminal cysteine rich region of syntaxin 11. These results suggest a pivotal role for S-acylation in the function of syntaxin 11 in NK cells

Syntaxin 11 recruits Munc18-2 to cytoplasmic membranes and to the activating immunological synapse.

<p>Analysis of the localization of Munc18-2 in YTS NK cells. (A) YTS cells were transfected with mCherry-Munc18-2, stained with LysoTracker Green to visualize secretory lysosomes and either imaged immediately (Resting) or conjugated with 721.221 target cells pre-stained with Cell Trace Far Red (blue in the merge image panels). (B) YTS cells were co-transfected with mCherry-Munc18-2 and GFP-syntaxin 11 and either imaged alone or after incubation with 721.221 cells pre-stained with Cell Trace Far Red. Cells were imaged using a Zeiss LSM700 laser scanning confocal microscope. Scale bars 5 µm.</p

The Q268X mutation abolishes membrane association of syntaxin 11 in YTS NK cells.

<p>(A) Analysis of the membrane association of syntaxin 11 expressed endogenously in YTS NK cells. Postnuclear supernatants from YTS NK cells were fractionated by centrifugation into pellet (membrane) and supernatant (cytosolic) fractions. The fractions were resolved by SDS-PAGE and immunoblots probed with antibodies specific for syntaxin 11, SNAP23, calnexin and GAPDH. (B) Analysis of the membrane association of GFP-syntaxin 11 and GFP-syntaxin 11 Q268X in YTS NK cells. Postnuclear supernatants of YTS cells transfected with either GFP-syntaxin 11 or GFP syntaxin 11 Q268X were fractionated by centrifugation into membrane and cytosolic fractions. The fractions were resolved by SDS-PAGE and GFP-syntaxin 11 fusion proteins detected by probing immunoblots with a rabbit syntaxin 11 specific antibody.</p

Analysis of the effect of FHL-4 mutations on the interaction of syntaxin 11 with SNAP23 and Munc18-2.

<p>(A) Co-immunopreciptation of syntaxin 11 and SNAP23. Prior to lysis, YTS NK cells were pre-incubated in the absence or presence of NEM. Cell lysates were then incubated in the presence or absence of a mouse syntaxin 11 specific antibody and antibody bound proteins pulled down with protein-G sepharose. The precipitated proteins were resolved by SDS-PAGE and immunoblots probed with SNAP23 and rabbit syntaxin 11 specific antibodies. (B) GST pulldowns with syntaxin 11 FHL-4 mutants. GST, GST-syntaxin 11 and GST fusions of FHL-4 mutants were bound to glutathione sepharose beads (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098900#pone.0098900.s001" target="_blank">Figure S1</a> for a coomassie stained gel of the GST fusions bound to the glutathione sepharose beads). Pulldowns of a cell lysate prepared from HeLa-M cells transfected with Myc-Munc18-2 were then performed with GST and the GST-fusions immobilized on glutathione sepharose. The precipitated proteins were resolved by SDS-PAGE and immunoblots probed with SNAP23 and Myc-tag specific antibodies.</p

Syntaxin 11 Q268X displays a diffuse cytosolic localisation in resting YTS NK cells and is not recruited to the activating immunological synapse.

<p>Analysis of the localization of wild type (A) syntaxin 11 and the Q268X mutant (B) in YTS NK cells. YTS cells transfected with either GFP-syntaxin 11 or GFP-syntaxin 11 Q268X were stained with LysoTracker Red to visualize secretory lysosomes and either imaged immediately (Resting) or conjugated to 721.221 target cells pre-stained with Cell Trace Far Red (blue in the merge image panels). Live cells were imaged using a Zeiss LSM700 laser scanning confocal microscope. Scale bars 5 µm.</p

The N-terminal 24 residues of syntaxin 11 are necessary for binding to Munc18-2 and for the stabilisation of syntaxin 11 expression by Munc18-2.

<p>(A) GST pulldowns with the syntaxin 11ΔN24 mutant. GST and GST-syntaxin 11ΔN24 were bound to glutathione sepharose (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098900#pone.0098900.s002" target="_blank">Figure S2</a> for a coomassie blue stained gel of the proteins bound to glutathione sepharose). Pulldowns of a cell lysate prepared from HeLa-M cells transfected with Myc-Munc18-2 were then performed with the GST and GST-syntaxin 11ΔN24 immobilized on glutathione sepharose The precipitated proteins were resolved by SDS-PAGE and analysed by immunoblotting with SNAP23 and Myc-tag specific antibodies. (B) Flow cytometric analysis of the effect of Munc18-2 on the expression of syntaxin 11. Constructs encoding GFP-syntaxin 11 or GFP-syntaxin 11ΔN24 were transfected into HeLa-M cells with either a control plasmid construct or a construct encoding Myc-Munc18-2. The mean fluorescence value for the cells was quantified by flow cytometry. Statistical analysis was performed using the Student's t-test. * P = 0.032. Error bars represent standard error of the mean for triplicate samples from 3 independent experiments. AU (arbitrary units). (C) Immunoblot analysis of the effect of Munc18-2 on the expression of syntaxin 11. Cell lysates prepared from the HeLa-M transfectants were analysed by immunoblotting with syntaxin 11, Myc-tag and GAPDH specific antibodies.</p

FHL-4 mutations highlight important functional regions of syntaxin 11.

<p>The N-terminal 24 residues of syntaxin 11 are required for binding to Munc18-2, the stabilization of syntaxin 11 expression by Munc18-2 and for the membrane recruitment of Munc18-2. The SNARE domain of syntaxin 11 is required for the interaction with the SNARE SNAP23. Cysteine residues within the C-terminal region are required for the S-acylation, membrane association and polarisation of syntaxin 11 to the activating immunological synapse in activated NK cells. The sites of each the FHL-4 mutations studied are indicated.</p

Cysteines in the C-terminal region of syntaxin 11 are required for membrane association.

<p>(A) C279, C280, C282, C283 and C285 (shown in red) were predicted by the CSS-PALM 2.04 software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098900#pone.0098900-Ren1" target="_blank">[38]</a> to be potential S-acylation sites in syntaxin 11. The corresponding 5 cysteine residues were mutated to alanine to generate the <i>de novo</i> mutant syntaxin 11C5A. (B) Analysis of the membrane association of syntaxin 11C5A in YTS NK cells. Postnuclear supernatants from YTS NK cells were fractionated by centrifugation into pellet (membrane) and supernatant (cytosolic) fractions. The fractions were resolved by SDS-PAGE and GFP-syntaxin 11C5A was detected by probing immunoblots with a syntaxin 11 specific antibody. (C) Analysis of the localization of the syntaxin 11C5A mutant in YTS NK cells. YTS cells expressing GFP-syntaxin 11C5A were then imaged in the absence of target cells (Resting) or conjugated to 721.221 cells pre-stained with Cell Trace (blue in the merge image panels). Cells were imaged using a Zeiss LSM700 laser scanning confocal microscope. Scale bars 5 µm.</p

Recruitment of Munc18-2 to cytoplasmic membranes and to the activating immunological synapse is dependent on the N- and C-terminal regions of syntaxin 11.

<p>Analysis of the effect of expression of the syntaxin 11ΔN24 and Q268X mutant proteins on the localization of Munc18-2 in YTS NK cells. mCherry-Munc18-2 was co-transfected with either GFP-syntaxin 11ΔN24 (A) or GFP-syntaxin 11 Q268X (B). YTS cells were then imaged in the absence of target cells (Resting) or conjugated to 721.221 target cells pre-stained with Cell Trace Far Red (blue in the merge image panels). Cells were imaged using a Zeiss LSM700 laser scanning confocal microscope. Scale bars 5 µm.</p

Syntaxin 11 is S-acylated and this is dependent on cysteines in the C-terminal region.

<p>(A) Acyl-biotinyl exchange. Free cysteines are blocked with the alkylating agent NEM, long chain fatty acid groups are cleaved from proteins using hydroxylamine revealing free cysteines, which are biotinoylated, enabling proteins to be pulled down with avidin beads. Non-transfected YTS cells (B) and YTS cells transfected with either GFP-syntaxin 11, GFP-syntaxin 11 Q268X or GFP-syntaxin 11C5A (C) were analysed with acyl-biotinyl exchange. Precipitated proteins from samples that had been incubated in the absence (negative control) or presence of hydroxylamine (S-acylated fraction) were resolved by SDS-PAGE. Syntaxin 11 and GFP-syntaxin 11 fusions were detected by probing immunoblots with syntaxin 11 specific antibodies. The non-S-acylated protein calreticulin served as a negative control and was detected with a rabbit anti-calreticulin antibody.</p