TAPL interaction network
The human lysosomal polypeptide ABC transporter TAPL (ABC subfamily B member 9, ABCB9) transports 6–59 amino-acids-long polypeptides from the cytosol into lysosomes. The subcellular localization of TAPL depends solely on its N-terminal transmembrane domain TMD0, which lacks conventional targeting sequences. However, the intracellular route and the molecular mechanisms that control TAPL localization remain unclear. Here, we delineated the route of TAPL to lysosomes and investigated the determinants of single trafficking steps. By synchronizing trafficking events by retention using selective hooks (RUSH) assay and visualizing individual intermediate steps through immunostaining and confocal microscopy, we demonstrate that TAPL takes the direct route to lysosomes. We further identified conserved charged residues within TMD0 transmembrane helices that are essential for individual steps of lysosomal targeting. Substitutions of these residues retained TAPL in the endoplasmic reticulum (ER) or Golgi. We also observed that for release from the ER, a salt bridge between Asp-17 and Arg-57 is essential. An interactome analysis revealed that Yip1-interacting factor homolog B, membrane-trafficking protein (YIF1B) interacts with TAPL. We also found that YIF1B is involved in ER-to-Golgi trafficking and interacts with TMD0 of TAPL via its transmembrane domain and that this interaction strongly depends on the newly identified salt bridge within TMD0. These results expand our knowledge about lysosomal trafficking of TAPL and the general function of extra transmembrane domains of ABC transporters.
Sample Processing Protocol
Anti-HA-immunoprecipitation was performed as previously described (Behrends et al, 2010; Jung et al, 2015; Jung et al, 2017; Sowa et al, 2009). Summarily, expression of TAPL-HA and coreTAPL-HA was induced by addition of 4 µg/ml doxycycline for 24 h in HeLa Flp-In T-REx cells. Parental non-transfected HeLa Flp-In T-REx cells were used as negative control. For each sample, 6.4 x 107 cells were harvested, frozen in liquid nitrogen and stored at -80 °C. Cells were lysed in 3 ml MCLB buffer (50 mM Tris, 150 mM NaCl, 0.5% NP40, pH 7.4) supplemented with cOmplete EDTA-free protease inhibitor tablets (Roche, 2 tablets for 50 ml MCLB buffer) on ice for 30 min. Lysate was cleared by centrifugation (18,000x g, 10 min, 4 °C) followed by filtering through a 0.45 µm spin filter (Millipore). For isolation of HA-tagged proteins, cell lysate was incubated overnight at 4 °C with 60 µl of equilibrated α-HA agarose beads (Sigma-Aldrich/Merck). Subsequently, beads were washed four times with 1 ml MCLB and 1 ml PBS, respectively. 50 µl of 250 µg/ml HA peptide was added to dry beads and incubated for 30 min at room temperature. Elution was repeated twice obtaining a final volume of 150 µl. Proteins were precipitated with 20% tri-chloroacetic acid (TCA), resuspended in 20 µl 50 mM ammonium bicarbonate pH 8.0 containing 10% acetonitrile and 750 ng trypsin (Promega) and incubated for 4 h at 37 °C. Desalting was performed using stage tips. Samples were analyzed as technical duplicates on an LTQ Velos (ThermoScientific).
Data Processing Protocol
Spectra were identified by Sequest searches as previously described (Huttlin et al, 2010). For CompPASS analysis, the identified peptides were compared to IP-MS data of 99 unrelated bait proteins, which were previously processed using the same experimental conditions (Sowa et al, 2009), to obtain weighted and normalized D-scores (WDN-score)(Table S4). Proteins with WDN ≥ 1.0 and APSM (average peptide spectral matches) ≥ 4 were considered as high-confident candidate interacting proteins (HCPs). To account for co-purifying (background) proteins in HeLa cells and coreTAPL-binding proteins, proteins found in these two IP conditions were subtracted from the list of TAPL HCIPs.