A Proteomic Analysis of Lysosomal Integral Membrane Proteins Reveals the Diverse Composition of the Organelle

Bagshaw, Richard D.; Mahuran, Don J.; Callahan, John W.

doi:10.1074/mcp.m400128-mcp200

Cited by 158 publications

(144 citation statements)

References 59 publications

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“…In contrast, LOPIT analyses the steady-state partitioning of proteins to establish their primary site of residency. A number of proteins previously reported as lysosomal (31,32) were identified in our analysis but did not cluster with the established lysosomal markers, indicating that they were not unique to the lysosome or predominantly localized there in DT40. Examples include nicastrin, a component of the gamma-secretase complex, which is located close to our PM cluster on PCA plots, and NiemannPick C1, found in an intermediate location close to the lysosome and Golgi clusters.…”

Section: Resultsmentioning

confidence: 72%

“…Rather than this being a problem with protein detection, we suggest that this reflects the steady-state protein distribution within the cell, and that there are actually relatively few membrane proteins uniquely localized to the lysosome. Although other proteomic studies focusing on lysosome membranes have reported a wide range of constituents (31,32), these have been based on the analysis of enriched lysosomal preparations and represent catalogues of all the proteins associated with the organelle. Included are residents of multiple compartments or proteins only transiently associated with the lysosome.…”

Section: Resultsmentioning

confidence: 99%

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The Organelle Proteome of the DT40 Lymphocyte Cell Line

Hall

Hester

Griffin

et al. 2009

Molecular & Cellular Proteomics

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A major challenge in eukaryotic cell biology is to understand the roles of individual proteins and the subcellular compartments in which they reside. Here, we use the localization of organelle proteins by isotope tagging technique to complete the first proteomic analysis of the major organelles of the DT40 lymphocyte cell line. This cell line is emerging as an important research tool because of the ease with which gene knockouts can be generated. We identify 1090 proteins through the analysis of preparations enriched for integral membrane or soluble and peripherally associated proteins and localize 223 proteins to the endoplasmic reticulum, Golgi, lysosome, mitochondrion, or plasma membrane by matching their density gradient distributions to those of known organelle residents. A striking finding is that within the secretory and The chicken pre-B cell line DT40 exhibits a remarkably high ratio of targeted to random integration for transfected DNA constructs. This property is unusual in vertebrate cell lines and enables targeted gene disruption experiments to be carried out with relative ease (1). Consequently, DT40 has become a major research tool for the molecular dissection of a wide range of cellular and biochemical mechanisms in a vertebrate context, including membrane traffic, signal transduction, and cell cycle (2).Proteins in eukaryotic cells are organized according to their functions within a dynamic network of membranes. Localization is therefore paramount in assigning functions to uncharacterized proteins and understanding the processes occurring in subcellular compartments. An increased knowledge of the protein localization within the DT40 cell line would be of great value. Traditional localization methods such as immunofluorescence microscopy are typically low throughput and are more suitably applied to the study of specific proteins of interest rather than the cataloguing of large numbers of proteins. Recent developments in proteomics have made it possible to analyze the protein composition of organelles using a variety of different approaches. Several groups have utilized label-free quantitative proteomics in the high throughput assignment of proteins to subcellular compartments. In one approach, protein correlation profiling, proteins from enriched organelle fractions are quantified by peptide ion intensity measurements (3, 4). Other similar methods employ quantitation by spectral counting, recording the number of ions detected per protein (5, 6). Localization of organelle proteins by isotope tagging (LOPIT) 1 is a complementary approach, which employs isotope labeling for quantitation (7-9). Rather than processing each sample separately as in label-free techniques, differentially labeled fractions are pooled early in the LOPIT protocol. This has the important advantage of reducing the points at which variation might be introduced into the data.LOPIT begins with the partial separation of organelles by density gradient centrifugation and relies on the assumption that proteins from each organelle c...

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Section: Resultsmentioning

confidence: 72%

Section: Resultsmentioning

confidence: 99%

The Organelle Proteome of the DT40 Lymphocyte Cell Line

Hall

Hester

Griffin

et al. 2009

Molecular & Cellular Proteomics

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“…Several mammalian ABC transporters localize to late endosomes/lysosomes, including the ABCB transporter (MDR/TAP) member 6, ABCA1, ABCA2, ABCA3, ABCA5, ABCB9 and the ABCC transporter MRP2 (Bagshaw et al, 2005;Chen et al, 2005;Cheong et al, 2006;Kubo et al, 2005;Neufeld et al, 2004;Zhang et al, 2000;Zhou et al, 2001). ABCA1 traffics between late endosomes and the cell surface and is required for the efflux from the cell of cholesterol in late endosomes (Chen et al, 2005;Neufeld et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Suppression of thecup-5mucolipidosis type IV-related lysosomal dysfunction by the inactivation of an ABC transporter inC. elegans

2006

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Mutations in MCOLN1, which encodes the protein mucolipin 1, result in the lysosomal storage disease mucolipidosis Type IV. Studies on human mucolipin 1 and on CUP-5, the Caenorhabditis elegans ortholog of mucolipin 1, have shown that these proteins are required for lysosome biogenesis/function. Loss of CUP-5 results in a defect in lysosomal degradation, leading to embryonic lethality. We have identified a mutation in the ABC transporter MRP-4 that rescues the degradation defect and the corresponding lethality, owing to the absence of CUP-5. MRP-4 localizes to endocytic compartments and its levels are elevated in the absence of CUP-5. These results indicate that the lysosomal degradation defect is exacerbated in some cells because of the accumulation of MRP-4 in lysosomes rather than the loss of CUP-5 per se. We also show that under some conditions, loss of MRP-4 rescues the embryonic lethality caused by the loss of the cathepsin L protease, indicating that the accumulation of ABC transporters may be a more general mechanism whereby an initial lysosomal dysfunction is more severely compromised.

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“…24,25 Acid phosphatases catalyze the hydrolysis of orthophosphate monoesters from a wide variety of substrates, including phosphorylated proteins, under acid conditions. 26,27 Several acid phosphatase (ACP) genes are known in mammals, of which ACP2 encodes the main lysosomal acid phosphatase.…”

Section: Cell Morphology and Cell Deathmentioning

confidence: 99%