Dataset Information

Targeting of non-phosphorylated lysosomal enzymes involves secretion and Lrp1-mediated recapture

ABSTRACT: Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types suggesting the existence of M6P-independent transport routes. SILAC-based analysis of purified lysosomes from M6P-deficient mouse fibroblasts (PTki) revealed unchanged amounts of 11 lysosomal enzymes, including cathepsin D and B (CtsD, CtsB), while demonstrating mossiorting of a variety of soluble lysosomal enzymes

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

DISEASE(S): Lysosomal Storage Disease

SUBMITTER: Melanie Thelen

LAB HEAD: Prof. Dr. Volkmar Gieselmann

PROVIDER: PXD001221 | Pride | 2015-05-01

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Markmannetal.2014Replicate1.msf	Msf
	Markmannetal.2014Replicate1.pep.xml	Pepxml
	Markmannetal.2014Replicate2.msf	Msf
	Markmannetal.2014Replicate2.pep.xml	Pepxml
	Markmannetal.2014Replicate3.msf	Msf

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Publications

Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting.

Markmann Sandra S Thelen Melanie M Cornils Kerstin K Schweizer Michaela M Brocke-Ahmadinejad Nahal N Willnow Thomas T Heeren Joerg J Gieselmann Volkmar V Braulke Thomas T Kollmann Katrin K

Traffic (Copenhagen, Denmark) 20150427 7

Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P residues results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types, suggesting the existence of M6P-independent transport routes. Here, we quantify the lysosomal proteome in M6P-deficient mouse fibroblasts (PT(ki)) using Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based ...[more]

PMID: 25786328

Similar Datasets

Project description:Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in parasitic protists. Whereas the mannose-6 phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide independent sorting was reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, protein targeting and involvement of lysosomes in hydrolase secretion. The organelles were purified using conventional Percoll and OptiPrep density gradient centrifugation, and a novel purification protocol based on phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in the lysosomal proteome of 462 proteins, which were sorted into 21 functional classes. Hydrolases represent the largest functional class and include proteases, lipases, phosphatases, glycosidases, and other hydrolases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate high dynamics of phagolysosomal compartment. Several lysosomal proteins including the cysteine protease TvCP2 were previously shown to be secreted. Our experiment showed that secretion of TvCP2 was strongly inhibited by chloroquine that increased intralysosomal pH and thus indicated TvCP2 secretion through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified in the phagolysosomal proteome divergent homologs of M6P receptor TvMPR, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP that possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as recognition marker for lysosomal hydrolases. Whether TvMPR or other possible receptors are involved in this process and what is the precise structure of the lysosomal recognition marker need to be clarified in future studies.