Abstract
Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions.
Highlights
IntroductionLysosomes are membrane-bound intracellular organelles that are key players in the degradation and recycling of biological material
Lysosomes are membrane-bound intracellular organelles that are key players in the degradation and recycling of biological material. Their crucial role in cell physiology is underlined by the existence of ϳ50 lysosomal storage diseases caused by genetic defects in lysosomal proteins or proteins involved in lysosome biogenesis [1]
The soluble content has first been analyzed by the use of an affinity purification protocol based on the mannose 6-phosphate modification (4 –11) that is characteristic of soluble lysosomal proteins [12]
Summary
Lysosomes are membrane-bound intracellular organelles that are key players in the degradation and recycling of biological material. The degradative function is carried out in the lysosomal lumen by the concerted action of over 60 hydrolases and accessory proteins [2] These soluble lysosomal proteins have been extensively studied, knowledge about membrane proteins remains rather limited, despite the multiple and crucial functions fulfilled by the membrane. It is responsible for establishing and maintaining pH and ionic gradients, transporting degradation substrates and products from/into the cytosol, and maintaining lysosome integrity. A comparative proteomics analysis based on spectral counts led to the selection of 734 candidate proteins They included on the one hand 94 novel potentially lysosomal proteins and, on the other hand, 46 established or putative transporters for which lysosomal residency is suggested by this study. We recently showed elsewhere that another candidate identified during this proteomic study, PQLC2, is a novel lysosomal amino acid transporter [36]
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