BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers

Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-SNARE VAMP7 mediates fusion of melanosomes with tubular transport carriers that also carry the cargo protein TYRP1 and that require BLOC-1 for their formation. Using live-cell imaging, we identify a pathway for VAMP7 recycling from melanosomes that employs distinct tubular carriers. The recycling carriers also harbor the VAMP7-binding scaffold protein VARP and the tissue-restricted Rab GTPase RAB38. Recycling carrier formation is dependent on the RAB38 exchange factor BLOC-3. Our data suggest that VAMP7 mediates fusion of BLOC-1–dependent transport carriers with melanosomes, illuminate SNARE recycling from melanosomes as a critical BLOC-3–dependent step, and likely explain the distinct hypopigmentation phenotypes associated with BLOC-1 and BLOC-3 deficiency in Hermansky–Pudlak syndrome variants.

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BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery

The Journal of Cell Biology ◽

10.1083/jcb.201410026 ◽

2015 ◽

Vol 209 (4) ◽

pp. 563-577 ◽

Cited By ~ 33

Author(s):

Megan K. Dennis ◽

Adriana R. Mantegazza ◽

Olivia L. Snir ◽

Danièle Tenza ◽

Amanda Acosta-Ruiz ◽

...

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Pigment Cell ◽

Wild Type ◽

Transport Pathway ◽

Cargo Transport ◽

Cargo Delivery ◽

Tubular Transport ◽

Hermansky Pudlak Syndrome ◽

Lysosome Related Organelles

Hermansky–Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2–deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2–deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation.

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SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models

Blood ◽

10.1182/blood-2011-11-389551 ◽

2012 ◽

Vol 120 (2) ◽

pp. 404-414 ◽

Cited By ~ 34

Author(s):

Ronghua Meng ◽

Yuhuan Wang ◽

Yu Yao ◽

Zhe Zhang ◽

Dawn C. Harper ◽

...

Keyword(s):

Platelet Activation ◽

Subcellular Fractionation ◽

Dense Granule ◽

Direct Role ◽

Excessive Bleeding ◽

Dense Granules ◽

Early Endosomes ◽

Hermansky Pudlak Syndrome ◽

Endosomal Transport ◽

Lysosome Related Organelles

Abstract Platelet dense granules are members of a family of tissue-specific, lysosome-related organelles that also includes melanosomes in melanocytes. Contents released from dense granules after platelet activation promote coagulation and hemostasis, and dense granule defects such as those seen in Hermansky-Pudlak syndrome (HPS) cause excessive bleeding, but little is known about how dense granules form in megakaryocytes (MKs). In the present study, we used SLC35D3, mutation of which causes a dense granule defect in mice, to show that early endosomes play a direct role in dense granule biogenesis. We show that SLC35D3 expression is up-regulated during mouse MK differentiation and is enriched in platelets. Using immunofluorescence and immunoelectron microscopy and subcellular fractionation in megakaryocytoid cells, we show that epitope-tagged and endogenous SLC35D3 localize predominantly to early endosomes but not to dense granule precursors. Nevertheless, SLC35D3 is depleted in mouse platelets from 2 of 3 HPS models and, when expressed ectopically in melanocytes, SLC35D3 localizes to melanosomes in a manner requiring a HPS-associated protein complex that functions from early endosomal transport intermediates. We conclude that SLC35D3 is either delivered to nascent dense granules from contiguous early endosomes as MKs mature or functions in dense granule biogenesis directly from early endosomes, suggesting that dense granules originate from early endosomes in MKs.

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BLOC-1 Is Required for Cargo-specific Sorting from Vacuolar Early Endosomes toward Lysosome-related Organelles

Molecular Biology of the Cell ◽

10.1091/mbc.e06-12-1066 ◽

2007 ◽

Vol 18 (3) ◽

pp. 768-780 ◽

Cited By ~ 139

Author(s):

Subba Rao Gangi Setty ◽

Danièle Tenza ◽

Steven T. Truschel ◽

Evelyn Chou ◽

Elena V. Sviderskaya ◽

...

Keyword(s):

Protein Complex ◽

Genetic Disorder ◽

Adaptor Protein ◽

Transport Pathways ◽

Cargo Transport ◽

Early Endosomes ◽

Hermansky Pudlak Syndrome ◽

Lysosome Related Organelles ◽

And Function ◽

Tyrosinase Related Protein

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1–deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS.

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The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER

The Journal of Cell Biology ◽

10.1083/jcb.200212101 ◽

2003 ◽

Vol 162 (3) ◽

pp. 403-412 ◽

Cited By ~ 47

Author(s):

Pierre Morsomme ◽

Cristina Prescianotto-Baschong ◽

Howard Riezman

Keyword(s):

Golgi Complex ◽

Protein Sorting ◽

Secretory Proteins ◽

Rab Gtpase ◽

Cargo Protein ◽

Conserved Oligomeric Golgi Complex ◽

Sorting Process ◽

Conserved Oligomeric Golgi

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1–1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1–1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

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Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

Journal of Cell Science ◽

10.1242/jcs.103.4.1139 ◽

1992 ◽

Vol 103 (4) ◽

pp. 1139-1152

Author(s):

J.W. Kok ◽

K. Hoekstra ◽

S. Eskelinen ◽

D. Hoekstra

Keyword(s):

Plasma Membrane ◽

Intracellular Ph ◽

Brefeldin A ◽

Fluid Phase ◽

Endocytic Pathway ◽

Late Endosomes ◽

Early Endosomes ◽

Recycling Pathway ◽

Golgi Network ◽

Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

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RABENOSYN separation-of-function mutations uncouple endosomal recycling from lysosomal degradation

10.1101/2021.10.03.21264281 ◽

2021 ◽

Author(s):

Franziska Paul ◽

Calista Ng ◽

Shahriar Nafissi ◽

Yalda Nilipoor ◽

Ali Reza Tavasoli ◽

...

Keyword(s):

Intellectual Disability ◽

Exome Sequencing ◽

Muscle Weakness ◽

Germline Mutations ◽

Lysosomal Degradation ◽

Mendelian Disorder ◽

Progressive Muscle Weakness ◽

Endosomal Recycling ◽

Early Endosomes ◽

Recycling Pathway

Rabenosyn (RBSN) is a conserved endosomal protein necessary for regulating internalized cargo. Here, we present genetic, cellular and biochemical evidence that two distinct RBSN missense variants are responsible for a novel Mendelian disorder consisting of progressive muscle weakness, facial dysmorphisms, ophthalmoplegia and intellectual disability. Using exome sequencing, we identified recessively-acting germline alleles p.Arg180Gly and p.Gly183Arg which are both situated in the FYVE domain of RBSN. We find that these variants abrogate binding to its cognate substrate PI3P and thus prevent its translocation to early endosomes. Although the endosomal recycling pathway was unaltered, mutant p.Gly183Arg patient fibroblasts exhibit accumulation of cargo tagged for lysosomal degradation. Our results suggest that these variants are separation-of-function alleles, which cause a delay in endosomal maturation without affecting cargo recycling. We conclude that distinct germline mutations in RBSN cause non-overlapping phenotypes with specific and discrete endolysosomal cellular defects.

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Mapping of Vps21 and HOPS Binding Sites in Vps8 and Effect of Binding Site Mutants on Endocytic Trafficking

Eukaryotic Cell ◽

10.1128/ec.00286-09 ◽

2010 ◽

Vol 9 (4) ◽

pp. 602-610 ◽

Cited By ~ 11

Author(s):

Agnes Pawelec ◽

Janja Arsić ◽

Ralf Kölling

Keyword(s):

Binding Sites ◽

Carboxypeptidase Y ◽

Rab Gtpase ◽

Physical Interaction ◽

Endosomal Trafficking ◽

Core Complex ◽

Content Type ◽

Multiple Contacts ◽

Late Endosomes ◽

Cargo Protein

ABSTRACT Vps8 is a subunit of the CORVET tethering complex, which is involved in early-to-late endosome fusion. Here, we examine the role of Vps8 in membrane fusion at late endosomes in Saccharomyces cerevisiae. We demonstrate that Vps8 associates with membranes and that this association is independent of the class C/HOPS core complex and, contrary to a previous report, also independent of the Rab GTPase Vps21. Our data indicate that Vps8 makes multiple contacts with membranes. One of these membrane binding regions could be mapped to the N-terminal part of the protein. By two-hybrid analysis, we obtained evidence for a physical interaction between Vps8 and the Rab5 homologue Vps21. In addition, the interaction with the HOPS core complex was confirmed by immunoprecipitation experiments. By deletion analysis, the Vps21 and HOPS binding sites were mapped in Vps8. Deletions that abrogated HOPS core complex binding had a strong effect on the turnover of the endocytic cargo protein Ste6 and on vacuolar sorting of carboxypeptidase Y. In contrast, deletions that abolished Vps21 binding showed only a modest effect. This suggests that the Vps21 interaction is not essential for endosomal trafficking but may be important for some other aspect of Vps8 function.

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