title: ErbB2-associated changes in the lysosomal proteome.
abstract: Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far depended on purification methods based on either density gradient centrifugations or magnetic purification of iron-loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar-type H(+) -ATPase. Quantitative MS-based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal membrane proteome was significantly altered by the ectopic expression of an active form of the ErbB2 oncogene, which renders the cells highly metastatic. The furthermost ErbB2-associated changes included increased levels of CD63, S100A11 and ferritin heavy chain. Overall, our data introduce the antibody-based purification of lysosomes as a suitable method for the characterization of lysosomes from a variety of pathological conditions with altered lysosomal density and stability.
authors.affiliation: Apoptosis Department and Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, Copenhagen, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Nylandsted
authors.forename: Andrea C
authors.initials: AC
authors.lastname: Becker
authors.forename: Jakob
authors.initials: J
authors.lastname: Bunkenborg
authors.forename: Jens S
authors.initials: JS
authors.lastname: Andersen
authors.forename: Jörn
authors.initials: J
authors.lastname: Dengjel
authors.forename: Marja
authors.initials: M
authors.lastname: Jäättelä
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D007425:Intracellular Membranes / Q000737:chemistry; D008247:Lysosomes / Q000737:chemistry* / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism
chemical_list: D020543:Proteome; D018719:Erb-b2 Receptor Tyrosine Kinases; D025262:Vacuolar Proton-Translocating ATPases; C508053:ERBB2 protein, human