Journal: The EMBO Journal
Article Title: GOLPH3 and GOLPH3L maintain Golgi localization of LYSET and a functional mannose 6-phosphate transport pathway
doi: 10.1038/s44318-024-00305-z
Figure Lengend Snippet: ( A , B ) Immunoblots demonstrating B4GALT5 hypersecretion from both LYSET and GNPTAB KO 293FT ( A ) (representative of two independent experiments) and HAP1 ( B ) cells. Conditioned media and membrane fractions were probed with the indicated antibodies. Calnexin was used as loading control for membrane fractions. ( C ) Ammonium chloride treatment leads to B4GALT5 hypersecretion. Conditioned media of WT and LYSET KO 293FT cells incubated overnight with or without 20 mM ammonium chloride were concentrated and subjected to immunoblotting. Enzymatic removal of N -glycans using PNGase F was performed where indicated. The electrophoretic mobility of the B4GALT5 proteoform secreted from WT cells (white arrowhead) was distinct from B4GALT5 secreted by LYSET KO cells (black arrowhead). This difference was abolished upon N -deglycosylation (gray arrowhead). One of two independent experiments is shown, and cathepsin L was used as control. Asterisks denote unspecific bands detected with the B4GALT5 antibodies. ( D ) The B4GALT5 signal detected in conditioned media from ammonium chloride-treated WT (white arrowhead) and both treated and untreated LYSET KO 293FT cells (black arrowhead) was absent from media of ammonium chloride-treated B4GALT5/B4GALT6 KO 293 cells, demonstrating the specificity of the antibodies used. Asterisks denote unspecific bands detected with the B4GALT5 antibodies. ( E ) Ammonium chloride-induced B4GALT5 secretion is dependent on SPPL3 activity. Immunoblots of conditioned media and membrane lysates of ammonium chloride-treated parental and SPPL3 KO Flp-In T-REx 293 cells as well as SPPL3 KO clones with doxycycline (dox) treatment (100 ng/ml for 48 h)-induced re-expression of SPPL3 WT and the active site mutant D271N. ( F ) M6P-affinity purification of ectopically expressed B4GALT5 in 293FT cells. WT, LYSET KO, or GNPTAB KO 293FT cells transfected with B4GALT5-V5 or B4GALT1-V5 expression constructs as indicated were treated with 10 mM ammonium chloride. Media, whole-cell lysates and eluates from anti-M6P immunoprecipitations were analyzed by immunoblot as indicated. ( G ) Maturation of a B4GALT5-mCherry RUSH reporter in WT and LYSET KO 293FT cells. Reporter release was induced by the treatment of cells with 50 µM D-biotin. Samples were collected at indicated time points and subjected to immunoblotting. Endogenous B4GALT1 (gray filled arrowheads), which is not M6P-tagged, was used as control. Filled black arrowheads denote the ectopically expressed B4GALT5 RUSH reporter and the open black arrowhead endogenous B4GALT5. Colored arrowheads illustrate the differential intracellular maturation of the B4GALT5 RUSH reporter: Blue arrowhead, not yet released, i.e., ER-resident B4GALT5; red arrowhead, B4GALT5 released from the ER in 293FT WT cells; green arrowhead, B4GALT5 released from the ER in LYSET KO cells carrying more extensive glycosylation. ( H ) Model explaining B4GALT5 secretion in LYSET KO cells. In WT cells, B4GALT5 is subject to M6P-tagging. SPPL3-mediated processing of transmembrane B4GALT5 into a soluble M6P-tagged protein enables its targeting to the endolysosome for degradation (left). Due to impaired M6P-tagging in LYSET KO cells, the SPPL3 cleavage product of B4GALT5 is not lysosomally targeted and secreted (center), which is abolished in LYSET- and SPPL3-double-deficient cells and may lead to intra-Golgi accumulation of B4GALT5 (right). .
Article Snippet: To generate a B4GALT1-V5 expression construct, cDNA encoding B4GALT1 (corresponding in sequence to NP_001488.2) was PCR-amplified from Str-KDEL-flGALT-SBP-tagBFP (Addgene item #65274) and cloned via HindIII and BamHI sites into a previously described pcDNA3.1-derived plasmid containing coding sequence for a C-terminal V5 tag (Hobohm et al, ).
Techniques: Western Blot, Membrane, Control, Incubation, Activity Assay, Clone Assay, Expressing, Mutagenesis, Affinity Purification, Transfection, Construct, Glycoproteomics
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