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Updated: Aug 16, 2026
Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics
Published on: June 23, 2022
Identification of novel lysosomal matrix proteins by proteome analysis
Katrin Kollmann1, Kudzai E Mutenda, Martina Balleininger
1Zentrum Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Georg-August Universität Göttingen, Göttingen, Germany.
Abstract:
The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.
Insights
Researchers identified new lysosomal matrix proteins using mannose 6-phosphate receptors (MPR) affinity purification. This study advances understanding of lysosomal protein targeting and function.
Area of Science:
- Cell Biology
- Proteomics
- Molecular Biology
Background:
- Lysosomal matrix proteins are crucial for lysosome function.
- Mannose 6-phosphate receptors (MPR) mediate the targeting of most lysosomal proteins.
- MPR-deficient cells secrete lysosomal proteins, offering a source for analysis.
Purpose of the Study:
- To comprehensively identify proteins that bind to MPRs.
- To discover novel lysosomal matrix proteins.
- To understand MPR-mediated protein trafficking to lysosomes.
Main Methods:
- Affinity purification of MPR-binding proteins from secretions of MPR-deficient mouse embryonic fibroblasts using MPR46 and MPR300 matrices.
- Identification of eluted proteins via mass spectrometry (2D-PAGE or MuDPIT).
- Verification of candidate protein binding and internalization via MPR-mediated endocytosis.
Main Results:
- Identified 34 known lysosomal matrix proteins, 4 candidate proteins, and 4 contaminants.
- Confirmed 3 candidate proteins (MERP-2, RISC, 66.3-kDa protein) bind to MPRs in a mannose 6-phosphate-dependent manner.
- Demonstrated MPR-mediated endocytosis and lysosomal targeting for these 3 candidate proteins.
Conclusions:
- Mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC), and a hypothetical 66.3-kDa protein are likely novel lysosomal matrix proteins.
- This study expands the known proteome of the lysosomal matrix.
- Provides insights into the mechanisms of lysosomal protein sorting and MPR function.
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