The First Transmembrane Domain of Lipid Phosphatase SAC1 Promotes Golgi Localization

<div><p>The lipid phosphatase Sac1 cycles between endoplasmic reticulum and cisternal Golgi compartments. In proliferating mammalian cells, a canonical dilysine motif at the C-terminus of Sac1 is required for coatomer complex-I (COP-I)-binding and continuous retrieval to the ER. Starvation triggers accumulation of Sac1 at the Golgi. The mechanism responsible for Golgi retention of Sac1 is unknown. Here we show that the first of the two transmembrane regions in human SAC1 (TM1) functions in Golgi localization. A minimal construct containing only TM1 and the adjacent flanking sequences is concentrated at the Golgi. Transplanting TM1 into transferrin receptor 2 (TfR2) induces Golgi accumulation of this normally plasma membrane and endosomal protein, indicating that TM1 is sufficient for Golgi localization. In addition, we determined that the N-terminal cytoplasmic domain of SAC1 also promotes Golgi localization, even when TM1 is mutated or absent. We conclude that the distribution of SAC1 within the Golgi is controlled via both passive membrane thickness-dependent partitioning of TM1 and a retention mechanism that requires the N-terminal cytoplasmic region.</p></div

The cytoplasmic flanking region of TM1 is required for ER export.

<p>HeLa cells were transfected with the indicated GFP-tagged SAC1 constructs (green), costained with anti-GM130 antibodies (red) or Sec61β (red) and analyzed by confocal immunofluorescence microscopy. (<b>A</b>) SAC1(478–549); (<b>B</b>) GFP-SAC1(478–543); (<b>C</b>) GFP-SAC1(501–549); (<b>D</b>) GFP-SAC1(512–549). Scale bar, 50 µm.</p

N-terminal domain of SAC1 promotes Golgi retention.

<p>HeLa cells were transfected with the indicated SAC1 constructs, costained with anti-GM130 antibodies (red) and analyzed by confocal immunofluorescence microscopy. (<b>A</b>) flag-SAC1(1–549)TMtfr2; (<b>B</b>) GFP-SAC1(TMtfr2)-K2A; <i>C</i>, GFP-SAC1ins3L-K2A. Scale bar, 50 µm.</p

Mapping the Golgi retention motif in SAC1.

<p>(<b>A</b>) Overview of truncated SAC1 constructs. (<b>B–G</b>) HeLa cells were transfected with the indicated FLAG-tagged (red) or GFP-tagged (green) SAC1 constructs, costained with anti-GM130 (red) or anti-GRASP65 antibodies (green) and analyzed by confocal immunofluorescence microscopy. (<b>B</b>) GFP-SAC1; (<b>C</b>) GFP-SAC1-K2A; (<b>D</b>) GFP-SAC1(1–518); (<b>E</b>) GFP-SAC1(478–587)-K2A; (<b>F</b>) FLAG-SAC1(1–549); (<b>G</b>) GFP-SAC1(478–549). Scale bar, 50 µm.</p

Lengthening TM1 by inserting three leucine residues results in a loss of Golgi retention.

<p>HeLa cells were transfected with the indicated GFP-tagged SAC1 constructs (green), costained with anti-GM130 antibodies (red) or WGA647 (red) and analyzed by confocal immunofluorescence microscopy. (<b>A</b>) GFP-SAC1(478–549); (<b>B</b>) GFP-SAC1(478–549)C2S; (<b>C</b>) GFP-SAC1(478–549)ins3L. Scale bar, 50 µm.</p

Insertion of TM1 induces Golgi retention of TfR2.

<p>HeLa cells were transfected with the indicated GFP-tagged TfR2 constructs that lack the N-terminal endocytic sorting motif (green), costained with anti-GM130 antibodies (red) or WGA647 (red) and analyzed by confocal immunofluorescence microscopy. (<b>A</b>) GFP-TfR2(73–801); (<b>B</b>) GFP-TfR2(73–801)TM1. Scale bar, 50 µm.</p

Temperature and pH profile of Coxyn A and Coxyl A.

<p><b>A</b> Effect of temperature on Coxyn A activity. <b>B</b> Effect of pH on Coxyn A activity. <b>C</b> Effect of temperature on Coxyl A activity. <b>D</b> Effect of pH on Coxyl A activity. <b>E</b> Effect of temperature on Coxyn A stability. <b>F</b> Effect of temperature on Coxyl A stability.</p

Hydrolysis of beechwood xylan at different concentration with constant loading of Xyn10A.

<p>A. TLC analysis of each hydrolysis products. B. The produced reducing sugar assay in each hydrolysis products. 0.1–2.0% (<i>w/v</i>) beechwood xylan was incubated with Xyn10A (0.5 µM, final concentration) at 80°C and pH 6.5 for 4 hours.</p

The phylogenetic trees of Coxyl A with similar enzymes.

<p><b>A</b> The relationship of Coxyl A (Δ) from <i>C. owensensis</i> with other similar GH39 xylosidases retrieved from NCBI database. <b>B</b> Sequence alignment of Coxyn A with those structure elucidated xylanses. The phylogenetic trees were constructed by MUSLE program and MEGA 6 Neighbor-joining analysis. Bootstrap values obtained with 1000 resamplings were indicated as percentages at all branches, and the scale bars represent 0.05 substitutions per amino acid position. Numbers followed by the names of the strains were accession numbers of NCBI.</p

Kinetic parameters of Coxyn A compared with other GH10 xylanases.

a<p>specific activity; BWX1: beechwood xylan; BWX2: birchwood xylan; OSX: oat splet xylan.</p