Cited by 459Cellular transport and secretionLysosomal Storage Disorders ResearchAutophagy in Disease and Therapy

The DOI matches its Crossref record; 48 of the 48 references it cites were found in a registry or an index; 14 of the 20 sources its claims cite were checked for support.

The sortilin cytoplasmic tail conveys Golgi–endosome transport and binds the VHS domain of the GGA2 sorting protein

Open access
Nielsen, Morten S., Madsen, Peder, Christensen, Erik I., Nykjær, Anders, Gliemann, Jørgen, Kasper, Dagmar, Pohlmann, Regina, Petersen, Claus M.·2001·The EMBO Journal

Abstract

Sortilin belongs to a growing family of multiligand type-1 receptors with homology to the yeast receptor Vps10p. Based on structural features and sortilin's intracellular predominance, we have proposed it to be a sorting receptor for ligands in the synthetic pathway as well as on the cell membrane. To test this hypothesis we examine here the cellular trafficking of chimeric receptors containing constructs of the sortilin tail. We report that sorting signals conforming to YXX and dileucine motifs mediate rapid endocytosis of sortilin chimeras, which subsequently travel to the trans-Golgi network, showing little or no recycling. Furthermore, we found that cation-independent mannose 6-phosphate receptor (MPR300)-sortilin chimeras, expressed in mannose 6-phosphate receptor knockout cells, were almost as efficient as MPR300 itself for transport of newly synthesized beta-hexosaminidase and beta-glucuronidase to lysosomes, and established that the sortilin tail contains potent signals for Golgi-endosome sorting. Finally, we provide evidence suggesting that sortilin is the first example of a mammalian receptor targeted by the recently described GGA family of cytosolic sorting proteins, which condition the Vps10p-mediated sorting of yeast carboxypeptidase Y.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

The sortilin cytoplasmic tail contains endocytic and Golgi–endosome sorting signals that direct chimeric receptors to the trans-Golgi network and can substitute for the MPR300 tail in lysosomal enzyme transport. Sortilin binds the GGA2 sorting protein via its VHS domain, and this interaction requires the C-terminal dileucine motif.

Key findings

  • Endocytosis of IL2R–sortilin chimeras depends mainly on the YXXF motif Y14SVL17 and, to a lesser extent, the C-terminal dileucine L51L52. At 30 min, non-internalized tracer was 41.5 ± 6.3% for wild-type tail, 76.5 ± 4.5% for Y14A/L17A, and 90.5 ± 1.2% for Y14A/L17A plus deletion of L51L52; combined disruption accounted for ~80% reduction in endocytosis.
  • Internalized IL2R–sortilin-wt traveled through clathrin-coated vesicles and Lamp-1-negative early endosomes to the TGN, co-localizing with TGN38 at 60 min, with little or no recycling over 4 h.
  • MPR300–sortilin-wt chimeras in mannose 6-phosphate receptor-deficient (mpr−) cells corrected secretion of newly synthesized β-hexosaminidase and β-glucuronidase and normalized lysosome morphology. At optimal expression, medium from transfected cultures contained ~80% less β-hexosaminidase than untransfected cells; with mannose 6-phosphate, <40% of the enzyme was found in the medium.
  • Sortilin binds GGA2 via the GGA2 VHS domain; >10% of the labeled sortilin pool was precipitated by GST–GGA2s, and deletion of the C-terminal dileucine abolished the interaction in two-hybrid and pull-down assays.

Methods

  • Chimeric receptors were generated by combining the sortilin cytoplasmic domain (wild-type or mutated/deleted at candidate motifs) with the luminal and transmembrane domains of IL2Rα (CD25/Tac) or MPR300. IL2R–sort chimeras were expressed in CHO-K1 cells; MPR300–sort chimeras were expressed in mouse embryonic fibroblasts deficient in both mannose 6-phosphate receptors (mpr−).
  • Endocytosis was measured with 125I-labeled anti-Tac/anti-CD25 and acid wash (pH 2.5); trafficking was analyzed by confocal and electron microscopy, surface biotinylation/metabolic labeling, and β-hexosaminidase fluorometric assays in the absence or presence of mannose 6-phosphate.
  • Protein interactions were tested by yeast two-hybrid screening with sortilin cytoplasmic domain as bait and by GST pull-down assays using GGA2 constructs, including the VHS domain and GAEH domain.

Limitations

  • The paper states that evidence for PACS-1 involvement and for the acidic cluster in sortilin trafficking was inconclusive; cell-to-cell variation was not insignificant and further evidence was needed. Full-length GGA2 could not be purified as a uniform protein.
  • Evident from the text: conclusions rely on chimeric receptors and antibody–receptor complexes, which may not fully reflect full-length sortilin behavior; several assays used transfected cells with varying expression levels and clone-to-clone differences.
  • No in vivo validation is reported.

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Integrity signals

Registry

Crossref · 10.1093/emboj/20.9.2180

Cites

48 of 48 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

45 Own result · 17 Own interpretation · 5 Supported · 5 Partly supported · 4 No full text of the sources · 3 Own method · accuracy not yet measured

Show all 43 claims

  • Probably not a substantial claim. 11
  • It may state two separate claims as one. 30
  • The quote only partly states this claim. 1
36 of 36
  • The paper relies on this without support.

    Mannose 6-phosphate inhibits ligand binding at the cell surface and prevents uptake from the medium.

    “Since mannose 6-phosphate inhibits ligand binding at the cell surface and thereby prevents uptake from the medium, it follows that the chimeric receptor, similarly to MPR300 itself, downregulates the cellular secretion of b-hexosaminidase.”
  • The paper relies on this without support.

    The MPR300-sort chimeras bind the mannose 6-phosphate moieties of proenzymes via the MPR300 luminal domain.

    “Since these chimeras (MPR300±sort) bind the mannose 6-phosphate moieties of proenzymes via the MPR300 luminal domain, we were then able to determine their sorting by monitoring the fate and cellular release of ligands, i.e. b-hexosaminidase.”
  • The paper relies on this without support.

    Sortilin is similar to the CPY sorting receptor Vps10p.

    “in view of the similarity between sortilin and the CPY sorting receptor Vps10p”
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Metadata

Authors
Nielsen, M. S., Madsen, P., Christensen, E. I., Nykjær, A., Gliemann, J., Kasper, D., Pohlmann, R., & Petersen, C. M.
Affiliations
  • Aarhus University (DK)
  • Universität Hamburg (DE)
  • University of Münster (DE)
Corresponding author
  • Nielsen, Morten S.
  • Petersen, Claus M.
Venue
The EMBO Journal
Published
2001-05-01
Volume
20
Issue
9
Pages
2180-2190
ISSN
  • 0261-4189 (ISSN-L)
  • 1460-2075
OpenAlex
W1997957892
Cited by
457
References
46
Citations by year
2026: 14 · 2025: 10 · 2024: 8 · 2023: 18 · 2022: 11 · 2021: 12 · 2020: 15 · 2019: 23 · 2018: 14 · 2017: 20 · 2016: 24 · 2015: 18 · 2014: 17 · 2013: 16 · 2012: 18
FWCI
6.89
Type
journal article
Language
en
Open access
Locations
  • The EMBO Journal (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Aarhus Universitet
  • Aarhus Universitets Forskningsfond
  • Novo Nordisk Fonden
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Health Sciences › Medicine › Genetics (clinical) › Lysosomal Storage Disorders Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
Keywords
sortilin, cytoplasmic tail, GGA2, VHS domain, endocytosis, YXXΦ motif, dileucine motif, trans-Golgi network, lysosomal sorting, mannose 6-phosphate receptor, beta-hexosaminidase, beta-glucuronidase, chimeric receptors, carboxypeptidase Y
MeSH terms
Sortilin, Amino Acid Sequence, Animals, Carrier Proteins, Cytoplasm, Endocytosis, Fungal Proteins, Golgi Apparatus, Cricetinae, Humans, Membrane Glycoproteins, Nerve Tissue Proteins, Protein Binding, Proteins, Receptors, Cell Surface, Endosomes, Recombinant Fusion Proteins, Transfection, Receptors, Interleukin-2, Gene Expression, CHO Cells, Protein Structure, Tertiary, Two-Hybrid System Techniques, Amino Acid Motifs, Protein Transport, Saccharomyces cerevisiae Proteins, Vesicular Transport Proteins, Adaptor Proteins, Vesicular Transport

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