The Phagosome Proteome

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Abstract

Phagosomes are key organelles for the innate ability of macrophages to participate in tissue remodeling, clear apoptotic cells, and restrict the spread of intracellular pathogens. To understand the functions of phagosomes, we initiated the systematic identification of their proteins. Using a proteomic approach, we identified >140 proteins associated with latex bead–containing phagosomes. Among these were hydrolases, proton pump ATPase subunits, and proteins of the fusion machinery, validating our approach. A series of unexpected proteins not previously described along the endocytic/phagocytic pathways were also identified, including the apoptotic proteins galectin3, Alix, and TRAIL, the anti-apoptotic protein 14-3-3, the lipid raft-enriched flotillin-1, the anti-microbial molecule lactadherin, and the small GTPase rab14. In addition, 24 spots from which the peptide masses could not be matched to entries in any database potentially represent new phagosomal proteins. The elaboration of a two-dimensional gel database of >160 identified spots allowed us to analyze how phagosome composition is modulated during phagolysosome biogenesis. Remarkably, during this process, hydrolases are not delivered in bulk to phagosomes, but are instead acquired sequentially. The systematic characterization of phagosome proteins provided new insights into phagosome functions and the protein or groups of proteins involved in and regulating these functions.

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Distinct Membrane Domains on Endosomes in the Recycling Pathway Visualized by Multicolor Imaging of Rab4, Rab5, and Rab11

Birte Sönnichsen, Stefano De Renzis, John Nielsen … (2000)

Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.

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Phase separation of integral membrane proteins in Triton X-114 solution.

C Bordier (1981)

A solution of the nonionic detergent Triton X-114 is homogeneous at 0 degrees C but separates in an aqueous phase and a detergent phase above 20 degrees C. The extent of this detergent phase separation increases with the temperature and is sensitive to the presence of other surfactants. The partition of proteins during phase separation in solutions of Triton X-114 is investigated. Hydrophilic proteins are found exclusively in the aqueous phase, and integral membrane proteins with an amphiphilic nature are recovered in the detergent phase. Triton X-114 is used to solubilize membranes and whole cells, and the soluble material is submitted to phase separation. Integral membrane proteins can thus be separated from hydrophilic proteins and identified as such in crude membrane or cellular detergent extracts.

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Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels.

A. Shevchenko, O Jensen, A. Podtelejnikov … (1996)

The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags. In blind trials, the method led to unambiguous identification in all cases. In the largest individual protein identification project to date, a total of 150 gel spots-many of them at subpicomole amounts-were successfully analyzed, greatly enlarging a yeast two-dimensional gel data base. More than 32 proteins were novel and matched to previously uncharacterized open reading frames in the yeast genome. This study establishes that mass spectrometry provides the required throughput, the certainty of identification, and the general applicability to serve as the method of choice to connect genome and proteome.

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Author and article information

Contributors

Département de pathologie et biologie cellulaire, Université de Montréal, C.P. 6128, Succ. Centre ville, Montréal, Québec, Canada, H3C 3J7.(514) 343-2459(514) 343-7250

Journal

Journal ID (nlm-ta): J Cell Biol

Title: The Journal of Cell Biology

Publisher: The Rockefeller University Press

ISSN (Print): 0021-9525

ISSN (Electronic): 1540-8140

Publication date (Print): 8 January 2001

Volume: 152

Issue: 1

Pages: 165-180

Affiliations

[a ]Laboratoire de Chimie des protéines, Commissariat a l'Energie Atomique, 38054 Grenoble, France

[b ]Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Quebec, Canada, H3C 3J7

[c ]Neurodégénérescence et Plasticité, Hopital A. Michallon, Centre Hospitalier Universitaire, 38043 Grenoble, France

Article

Publisher ID: 0009065

DOI: 10.1083/jcb.152.1.165

PMC ID: 2193653

PubMed ID: 11149929

SO-VID: 48e25cff-f9b8-4b0e-9acc-09a490a72353

History

Date received : 15 September 2000

Date revision requested : 9 November 2000

Date accepted : 10 November 2000

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