title: Attachment and fusion of endoplasmic reticulum with vacuoles containing Legionella pneumophila.
abstract: Legionella pneumophila is an intracellular pathogen that replicates in a unique vacuole that avoids endocytic maturation. Previous studies have shown host vesicles attached to the L. pneumophila-containing vacuole (LCV) minutes after uptake. Here we examine the origin and content of these vesicles by electron microscopy (EM). Our data demonstrate that the attached vesicles are derived from endoplasmic reticulum (ER) based the presence of the resident ER proteins glucose-6-phosphatase, protein disulphide isomerase (PDI) and proteins having the ER-retention signal lysine-aspartic acid-glutamic acid-leucine (KDEL). After tethering occurred, ER markers inside of attached vesicles were delivered into the lumen of the LCV, indicating ER fusion. Treatment of cells with brefeldin A did not interfere with the attachment of ER vesicles with the LCV, suggesting that tethering of these vesicles does not require activities mediated by ADP-ribosylation factor (ARF). ER vesicles were not tethered to the LCV in cells producing the Sar1H79G protein, indicating that vesicles produced by the Sar1/CopII system are necessary for vesicle attachment. From these data we conclude that formation of the organelle that supports L. pneumophila replication is a two-stage process that involves remodelling of the LCV by early secretory vesicles produced by the Sar1/CopII system, followed by attachment and fusion of ER.
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.
authors.forename: Camenzind G
authors.initials: CG
authors.lastname: Robinson
authors.forename: Craig R
authors.initials: CR
authors.lastname: Roy
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000382:microbiology / Q000502:physiology* / Q000648:ultrastructure; D005952:Glucose-6-Phosphatase / Q000378:metabolism; D016952:Legionella pneumophila / Q000502:physiology*; D008264:Macrophages / Q000382:microbiology / Q000502:physiology* / Q000648:ultrastructure; D008561:Membrane Fusion; D051379:Mice; D046529:Microscopy, Electron, Transmission; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014617:Vacuoles / Q000382:microbiology / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D015415:Biomarkers; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D005952:Glucose-6-Phosphatase; D020727:ADP-Ribosylation Factors