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Lysosomal proteomics by affinity purification-mass spectrometry

ABSTRACT: The aims of this project is to establish a comprehensive proteomic map of the lysosomes using isolated lysosomes via affinity purification. Affinity-purified mitochondria was used as a control to demonstrate the organelle specificity. In this study, we identified several lipid transporters that are associated with the lysosome and further characterized their roles in sterol-dependent mTORC1 regulation.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Chun-Yan Lim

LAB HEAD: Roberto Zoncu

PROVIDER: PXD014733 | Pride | 2019-09-27

REPOSITORIES: Pride

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			Action	DRS
	170706_32_66roberto_A1.mzid.gz	Mzid
	170706_32_66roberto_A1.mzid_170706_32_66roberto_A1.MGF	Mzid
	170706_32_66roberto_A1.mzid_170706_32_66roberto_A1.MGFcopy	Mzid
	170706_32_66roberto_A1.mzid_170706_32_66roberto_A1.pride.mgf.gz	Mzid
	170706_32_66roberto_A1.pride.mztab.gz	Mztab

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Publications

ER-lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann-Pick type C.

Lim Chun-Yan CY Davis Oliver B OB Shin Hijai R HR Zhang Justin J Berdan Charles A CA Jiang Xuntian X Counihan Jessica L JL Ory Daniel S DS Nomura Daniel K DK Zoncu Roberto R

Nature cell biology 20190923 10

Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes by the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signalling are unknown. Here, we show that oxysterol binding protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA and VAPB, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-in ...[more]

PMID: 31548609

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A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

Project description:Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.

2020-10-29 | PXD021444 | Pride

Identification and characterization of a ribosome receptor for starvation-induced ribophagy

Project description:The lysosome degrades and recycles macromolecules, signals to the master growth regulator mTORC1, and is associated with human disease. Here, we performed quantitative proteomic analyses of lysosomes rapidly isolated using the LysoIP method and find that nutrient levels and mTOR dynamically modulate the lysosomal proteome. We focus on NUFIP1, a protein that upon mTORC1 inhibition redistributes from the nucleus to autophagosomes and lysosomes. Upon these conditions, NUFIP1 interacts with ribosomes and delivers them to autophagosomes by directly binding to LC3B. The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. We conclude that NUFIP1 is a receptor for the selective autophagy of ribosomes.

2018-06-21 | PXD009084 | Pride

MFSD1 KO Liver - Lysosomes are a major site of intracellular acidic hydrolase-mediated proteolysis and cellular degradation

Project description:Lysosomes are a major site of intracellular acidic hydrolase-mediated proteolysis and cellular degradation. The export of low-molecular catabolic end products from the lysosomal lumen to the cytosol is facilitated by polytopic transmembrane proteins which mediate secondary active or passive transport. One of these proteins is MFSD1, a so far uncharacterized lysosomal 12 transmembrane domain-containing family member of the major facilitator superfamily. MFSD1 is, uncommon for a lysosomal membrane protein, not N-glycosylated. It contains a dileucine-based sorting motif that is essential for its transport to lysosomes. Lysosomes from mouse liver of MFSD1 knock-out mice were isolated and analyzed by TMT labelling, as well as whole liver protein extracts, both in comparison to wildtype controls.

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The faint sausage gene encodes a subunit of the spliceosome-activating Prp19 complex and is required for tracheal branching morphogenesis in Drosophila

Project description:Morphogenesis requires the dynamic regulation of gene expression, including transcription, mRNA maturation and translation. Dysfunction of general components of the splicing machinery can cause surprisingly specific phenotypes, but the basis for these cell-type specific effects is not clear. Here we show that the faint sausage (fas) locus, implicated in epithelial morphogenesis and previously reported to encode a secreted immunoglobulin domain protein, in fact encodes a subunit of the Prp19 complex that is essential for efficient pre-mRNA splicing. Loss of zygotic fas function impairs the efficiency of splicing, associated with widespread retention of introns in mature mRNAs and dramatic changes in gene expression. Surprisingly, despite these general effects, zygotic fas mutants show specific defects in tracheal cell migration. Zygotic fas function becomes essential during late embryogenesis when maternally supplied splicing factors decline. We propose that tracheal branching, which relies on dynamic changes in gene expression, is particularly sensitive for efficient spliceosome function. Our results provide an entry point to study functions of splicing during organogenesis and provide a better understanding of specific disease phenotypes associated with mutations in general splicing factors.

2016-11-18 | E-MTAB-5069 | biostudies-arrayexpress

LYSET/TMEM251 KO cellular and lysosomal proteomes from MIA PaCa-2 cells

Project description:Proteome analysis of post-nuclear supernatants (PNS) and enriched lysosomal fractions from LYSET/TMEM251 knockout and control MIA PaCa-2 cells. Lysosomes were loaded with iron nanoparticles (DexoMAG C, Liquids Research). PNS were generated from cell homogenates and used as input to enrich lysosomal fractions over magnetic LS MACS columns (Miltenyi).

2022-09-29 | PXD029659 | Pride

ChIP-seq analysis of SREB during yeast phase growth of Blastomyces dermatitidis at 37oC

Project description:ChIP-seq analysis was used to identify B. dermatitidis genes bound by the GATA transcription factor encoded by SREB during growth as yeast at 37oC SREB was engineered to contain an in-frame 3x-hemagglutinin (HA) epitope tag at the C-terminus. The SREB-3xHA construct was under control of its native promoter and contained the 3-untranslated region. Using Agrobacterium tumefaciens, B. dermatitidis ATCC 26199 was transformed with the SREB-3xHA construct (referred to a SREB-3xHA strain in this document). The SREB-3xHA construct was functional because retransformation of SREB? with the construct complented the null mutant. Chromatin was extracted and sheared from ATCC 26199 and SREB-3xHA yeast grown in liquid Histoplasma macrophage medium (HMM) containing 10 ?M iron sulfate (FeSO4) at 37oC. ATCC 26199 was the untagged control strain.

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The Legionella protein VpdC is a ubiquitin-activated phospholipase effector

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2023-06-12 | PXD037059 | Pride

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

Project description: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

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Male Rat DNA methylation in the Prefrontal Cortex at postnatal day 62 following prenatal restraint stress

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Project description:Heavy metal-resistant bacteria secrete extracellular proteins (e-PNs). However, the role of e-PNs in heavy metal resistance remains elusive. Here Fourier Transform Infrared Spectroscopy implied that N-H, C = O and NH2-R played a crucial role in the adsorption and resistance of Ni2+ in the model organism Cuprividus pauculus 1490 (C. pauculus). Proteinase K treatment reduced Ni2+ resistance of C. pauculus underlining the essential role of e-PNs. Further three-dimension excitation-emission matrix fluorescence spectroscopy analysis demonstrated that tryptophan proteins as part of the e-PNs increased significantly with Ni2+ treatment. Proteomic and quantitative real-time polymerase chain reaction data indicated that major changes were induced in the metabolism of C. pauculus in response to Ni2+. Among those lipopolysaccharide biosynthesis, general secretion pathways, Ni2+-affiliated transporters and multidrug efflux play an essential role in Ni2+ resistance. Altogether the results provide a conceptual model for comprehending how e-PNs contribute to bacterial resistance and adsorption of Ni2+.

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