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GOLPH3 and GOLPH3L maintain Golgi localization of LYSET and a functional mannose 6-phosphate transport pathway

ABSTRACT: Glycosylation, which plays an important role in modifying lipids and sorting of proteins, is regulated by asymmetric intra-Golgi distribution and SPPL3-mediated cleavage of Golgi enzymes. We found that cells lacking LYSET/TMEM251, a retention factor for Golgi N-acetylglucosamine-1-phosphotransferase (GNPT), display SPPL3-dependent hypersecretion of the Golgi membrane protein B4GALT5. We demonstrate that in wild-type cells B4GALT5 is tagged with mannose 6-phosphate (M6P), a sorting tag typical of soluble lysosomal hydrolases Hence, M6P-tagging of B4GALT5 may represent a novel degradative lysosomal pathway. We also observed B4GALT5 hypersecretion and prominent destabilization of LYSET-GNPT complexes, impaired M6P-tagging and disturbed maturation and trafficking of lysosomal enzymes in multiple human cell lines lacking the COPI adaptors GOLPH3 and GOLPH3L. Mechanistically, we identified LYSET as a novel, atypical client of GOLPH3/GOLPH3L. Thus, by ensuring the cis-Golgi localization of the LYSET-GNPT complex and maintaining its Golgi polarity, GOLPH3/GOLPH3L is essential for the integrity of the M6P-tagging machinery and homeostasis of lysosomes.

SUBMITTER: Ms. Berit, K. Brauer

PROVIDER: S-SCDT-10_1038-S44318-024-00305-Z | biostudies-other |

REPOSITORIES: biostudies-other

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GOLPH3 and GOLPH3L maintain Golgi localization of LYSET and a functional mannose 6-phosphate transport pathway

Project description:Glycosylation plays an important role for modifications of lipids and sorting of proteins that is regulated by asymmetric intra-Golgi distribution and SPPL3-mediated cleavage of Golgi enzymes. We found that cells lacking the Golgi N-acetylglucosamine phosphotransferase (GNPT) retention factor LYSET/TMEM251 display a SPPL3-dependent hypersecretion of the galactosyltransferase B4GALT5 which is caused by the loss of its lysosomal mannose 6-phosphate (M6P) targeting signal. Similarly, loss of the COPI adaptors GOLPH3/GOLPH3L destabilized LYSET-GNPT complexes and led to a prominent hypersecretion of B4GALT5 and lysosomal enzymes. Mechanistically, we identified LYSET as a novel, atypical client of GOLPH3/GOLPH3L. GOLPH3/GOLPH3L hence ensure cis-Golgi localization of the LYSET-GNPT complex and maintain Golgi polarity, integrity of the M6P tagging machinery and homeostasis of lysosomes.

2024-11-27 | PXD052083 | Pride

Transport of mannose-6-phosphate receptors from the trans-Golgi network to endosomes requires Rab31.

Project description:Rab31, a protein that we originally cloned from a rat oligodendrocyte cDNA library, localizes in the trans-Golgi network (TGN) and endosomes. However, its function has not yet been established. Here we show the involvement of Rab31 in the transport of mannose 6-phosphate receptors (MPRs) from TGN to endosomes. We demonstrate the specific sorting of cation-dependent-MPR (CD-MPR), but not CD63 and vesicular stomatitis virus G (VSVG) protein, to Rab31-containing trans-Golgi network carriers. CD-MPR and Rab31 containing carriers originate from extending TGN tubules that also contain clathrin and GGA1 coats. Expression of constitutively active Rab31 reduced the content of CD-MPR in the TGN relative to that of endosomes, while expression of dominant negative Rab31 triggered reciprocal changes in CD-MPR distribution. Expression of dominant negative Rab31 also inhibited the formation of carriers containing CD-MPR in the TGN, without affecting the exit of VSVG from this compartment. Importantly, siRNA-mediated depletion of endogenous Rab31 caused the collapse of the Golgi apparatus. Our observations demonstrate that Rab31 is required for transport of MPRs from TGN to endosomes and for the Golgi/TGN organization.

| S-EPMC2753287 | biostudies-literature

ArfGAP3 regulates the transport of cation-independent mannose 6-phosphate receptor in the post-Golgi compartment.

Project description:ArfGAPs are known to be involved in cargo sorting in COPI transport. However, the role of ArfGAPs in post-Golgi membrane traffic has not been defined. To determine the function of ArfGAPs in post-Golgi traffic, we used small interfering RNA to examine each of 25 ArfGAPs for effects on cation-independent mannose 6-phosphate receptor (CIMPR) localization. We found that downregulation of ArfGAP3 resulted in the peripheral localization of CIMPR. The effect was specific for ArfGAP3 and dependent on its GAP activity, because the phenotype was rescued by ArfGAP3 but not by ArfGAP1, ArfGAP2, or the GAP domain mutants of ArfGAP3. ArfGAP3 localized to the trans-Golgi network and early endosomes. In cells with reduced expression of ArfGAP3, Cathepsin D maturation was slowed and its secretion was accelerated. Also retrograde transport from the endosomes to the trans-Golgi network of endogenous CIMPR, but not truncated CIMPR lacking the luminal domain, was perturbed in cells with reduced expression of ArfGAP3. Furthermore the exit of epidermal growth factor receptor (EGFR) from the early endosomes and degradation of EGFR after EGF stimulation was slowed in cells with reduced expression of ArfGAP3. ArfGAP3 associates with Golgi-localized, γ-ear-containing, ADP-ribosylation factor binding proteins (GGAs), and ArfGAP3 knockdown reduces membrane association of GGAs. A possible mechanism explaining our results is that ArfGAP3 regulates transport from early endosomes to late endosomes. We suggest a model in which ArfGAP3 regulates Golgi association of GGA clathrin adaptors.

| S-EPMC3795807 | biostudies-literature

GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway

Project description:To investigate the expression profilings after knocking out TMEM251. We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 different TMEM251 sgRNA knockout cells compared to WT

2022-07-28 | GSE209652 | GEO

Biochemical characterization and lysosomal localization of the mannose-6-phosphate protein p76 (hypothetical protein LOC196463).

Project description:Most soluble lysosomal proteins carry Man6P (mannose 6-phosphate), a specific carbohydrate marker that enables their binding to cellular MPRs (Man6P receptors) and their subsequent targeting towards the lysosome. This characteristic was exploited to identify novel soluble lysosomal proteins by proteomic analysis of Man6P proteins purified from a human cell line. Among the proteins identified during the course of the latter study [Journet, Chapel, Kieffer, Roux and Garin (2002) Proteomics, 2, 1026-1040], some had not been previously described as lysosomal proteins. We focused on a protein detected at 76 kDa by SDS/PAGE. We named this protein 'p76' and it appeared later in the NCBI protein database as the 'hypothetical protein LOC196463'. In the present paper, we describe the identification of p76 by MS and we analyse several of its biochemical characteristics. The presence of Man6P sugars was confirmed by an MPR overlay experiment, which showed the direct and Man6P-dependent interaction between p76 and the MPR. The presence of six N-glycosylation sites was validated by progressive peptide-N-glycosidase F deglycosylation. Experiments using N- and C-termini directed anti-p76 antibodies provided insights into p76 maturation. Most importantly, we were able to demonstrate the lysosomal localization of this protein, which was initially suggested by its Man6P tags, by both immunofluorescence and sub-cellular fractionation of mouse liver homogenates.

| S-EPMC1863569 | biostudies-literature

GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway.

Project description:The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discover TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 causes mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrate that TMEM251 localizes to the Golgi and is required for the cleavage and activity of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 as GNPTAB cleavage and activity factor (GCAF) and its related disease as Mucolipidosis Type V.

| S-EPMC9468337 | biostudies-literature

Mannose 6-phosphate receptor homology (MRH) domain-containing lectins in the secretory pathway.

Project description:BackgroundThe mannose 6-phosphate receptor homology (MRH) domain-containing family of proteins, which include recycling receptors (mannose 6-phosphate receptors, MPRs), resident endoplasmic reticulum (ER) proteins (glucosidase II β-subunit, XTP3-B, OS-9), and a Golgi glycosyltransferase (GlcNAc-phosphotransferase γ-subunit), are characterized by the presence of one or more MRH domains. Many MRH domains act as lectins and bind specific phosphorylated (MPRs) or non-phosphorylated (glucosidase II β-subunit, XTP3-B and OS-9) high mannose-type N-glycans. The MPRs are the only proteins known to bind mannose 6-phosphate (Man-6-P) residues via their MRH domains.Scope of reviewRecent biochemical and structural studies that have provided valuable insight into the glycan specificity and mechanisms of carbohydrate recognition by this diverse group of MRH domain-containing proteins are highlighted.Major conclusionsCurrently, three-dimensional structures are known for ten MRH domains, revealing the conservation of a similar fold. OS-9 and the MPRs use the same four residues (Gln, Arg, Glu, and Tyr) to bind mannose.General significanceThe MRH domain-containing proteins play key roles in the secretory pathway: glucosidase II, XTP3-B, and OS-9 are involved in the recognition of nascent glycoproteins, whereas the MPRs play an essential role in lysosome biogenesis by targeting Man-6-P-containing lysosomal enzymes to the lysosome.

| S-EPMC3150748 | biostudies-literature

Spatial regulation of Golgi phosphatidylinositol-4-phosphate is required for enzyme localization and glycosylation fidelity.

Project description:The enrichment of phosphatidylinositol-4-phosphate (PI(4)P) at the trans Golgi network (TGN) is instrumental for proper protein and lipid sorting, yet how the restricted distribution of PI(4)P is achieved remains unknown. Here, we show that lipid phosphatase Suppressor of actin mutations 1 (SAC1) is crucial for the spatial regulation of Golgi PI(4)P. Ultrastructural analysis revealed that SAC1 is predominantly located at cisternal Golgi membranes but is absent from the TGN, thus confining PI(4)P to the TGN. RNAi-mediated knockdown of SAC1 caused changes in Golgi morphology and mislocalization of Golgi enzymes. Enzymes involved in glycan processing such as mannosidase-II (Man-II) and N-acetylglucosamine transferase-I (GnT-I) redistributed to aberrant intracellular structures and to the cell surface in SAC1 knockdown cells. SAC1 depletion also induced a unique pattern of Golgi-specific defects in N-and O-linked glycosylation. These results indicate that SAC1 organizes PI(4)P distribution between the Golgi complex and the TGN, which is instrumental for resident enzyme partitioning and Golgi morphology.

| S-EPMC2921771 | biostudies-literature

A functional role for the GCC185 golgin in mannose 6-phosphate receptor recycling.

Project description:Mannose 6-phosphate receptors (MPRs) deliver newly synthesized lysosomal enzymes to endosomes and then recycle to the Golgi. MPR recycling requires Rab9 GTPase; Rab9 recruits the cytosolic adaptor TIP47 and enhances its ability to bind to MPR cytoplasmic domains during transport vesicle formation. Rab9-bearing vesicles then fuse with the trans-Golgi network (TGN) in living cells, but nothing is known about how these vesicles identify and dock with their target. We show here that GCC185, a member of the Golgin family of putative tethering proteins, is a Rab9 effector that is required for MPR recycling from endosomes to the TGN in living cells, and in vitro. GCC185 does not rely on Rab9 for its TGN localization; depletion of GCC185 slightly alters the Golgi ribbon but does not interfere with Golgi function. Loss of GCC185 triggers enhanced degradation of mannose 6-phosphate receptors and enhanced secretion of hexosaminidase. These data assign a specific pathway to an interesting, TGN-localized protein and suggest that GCC185 may participate in the docking of late endosome-derived, Rab9-bearing transport vesicles at the TGN.

| S-EPMC1635343 | biostudies-literature

A role of Rab29 in the integrity of the trans-Golgi network and retrograde trafficking of mannose-6-phosphate receptor.

Project description:Rab29 (also referred as Rab7L1) is a novel Rab protein, and is recently demonstrated to regulate phagocytosis and traffic from the Golgi to the lysosome. However, its roles in membrane trafficking have not been investigated extensively. Our results in this study revealed that Rab29 is associated with the trans-Golgi network (TGN), and is essential for maintaining the integrity of the TGN, because inhibition of the activity of Rab29 or depletion of Rab29 resulted in fragmentation of the TGN marked by TGN46. Expression of the dominant negative form Rab29T21N or shRNA-Rab29 also altered the distribution of mannose-6-phosphate receptor (M6PR), and interrupted the retrograde trafficking of M6PR through monitoring the endocytosis of CD8-tagged calcium dependent M6PR (cdM6PR) or calcium independent M6PR (ciM6PR), but without significant effects on the anterograde trafficking of vesicular stomatitis virus G protein (VSV-G). Our results suggest that Rab29 is essential for the integrity of the TGN and participates in the retrograde trafficking of M6PRs.

| S-EPMC4008501 | biostudies-literature

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