Organelle proteomics: identification of the exocytic machinery associated with the natural killer cell secretory lysosome.
Natural killer (NK) cells and cytotoxic T lymphocytes eliminate virally infected and transformed cells. Target cell killing is mediated by the regulated exocytosis of secretory lysosomes, which deliver perforin and proapoptotic granzymes to the infected or transformed cell. Yet despite the central role that secretory lysosome exocytosis plays in the immune response to viruses and tumors, little is known about the molecular machinery that regulates the docking and fusion of this organelle with the plasma membrane. To identify potential components of this exocytic machinery we used proteomics to define the protein composition of the NK cell secretory lysosome membrane. Secretory lysosomes were isolated from the NK cell line YTS by subcellular fractionation, integral membrane proteins and membrane-associated proteins were enriched using Triton X-114 and separated by SDS-PAGE, and tryptic peptides were identified by LC ESI-MS/MS. In total 221 proteins were identified unambiguously in the secretory lysosome membrane fraction of which 61% were predicted to be either integral membrane proteins or membrane-associated proteins. A significant proportion of the proteins identified play a role in vesicular trafficking, including members of both the Rab GTPase and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and protein families. These proteins include Rab27a and the SNARE vesicle-associated membrane protein-7, both of which were enriched in the secretory lysosome fraction and represent potential components of the machinery that regulates the exocytosis of this organelle in NK cells.
Casey
Tammy M
TM
Institute of Molecular and Cellular Biology, Garstang Building, University of Leeds, Leeds LS2 9JT, UK.
Meade
Josephine L
JL
Hewitt
Eric W
EW
D002460:Cell Line; D002853:Chromatography, Liquid; D005089:Exocytosis; D007694:Killer Cells, Natural / Q000737:chemistry* / Q000166:cytology; D008247:Lysosomes / Q000737:chemistry*; D040901:Proteomics; D050683:R-SNARE Proteins / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D020691:rab GTP-Binding Proteins / Q000032:analysis; D000075745:rab27 GTP-Binding Proteins
D050683:R-SNARE Proteins; C121241:VAMP7 protein, human; D000075745:rab27 GTP-Binding Proteins; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins