TY - JOUR

T1 - ErbB2-associated changes in the lysosomal proteome

AU - Nylandsted, Jesper

AU - Becker, Andrea C

AU - Bunkenborg, Jakob

AU - Andersen, Jens S

AU - Dengjel, Jörn

AU - Jaattela, Marja

N1 - Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PY - 2011

Y1 - 2011

N2 - 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.

AB - 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.

KW - Cell Line, Tumor

KW - Humans

KW - Immunomagnetic Separation

KW - Intracellular Membranes

KW - Lysosomes

KW - Proteome

KW - Proteomics

KW - Receptor, erbB-2

KW - Vacuolar Proton-Translocating ATPases

U2 - 10.1002/pmic.201000734

DO - 10.1002/pmic.201000734

M3 - Journal article

C2 - 21674799

VL - 11

SP - 2830

EP - 2838

JO - Proteomics - Practical Proteomics

JF - Proteomics - Practical Proteomics

SN - 1862-7595

IS - 14

ER -