55 research outputs found
Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells.
The Glyceraldehyde-3-Phosphate Dehydrogenase and the Small GTPase Rab 2 Are Crucial for Brucella Replication
The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the “Brucella-containing vacuole” (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC ι, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells
Structural insight into the membrane targeting domain of the Legionella deAMPylase SidD
AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus
far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also
encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle
transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like
domain of SidD. Here, we determined the crystal structure of full length SidD including the
uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within
the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus
within mammalian cells. Deletion of the loop (??loop) or substitution of its aromatic phenylalanine
residues rendered SidD cytosolic, showing that the hydrophobic loop is the
primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal
alpha helices resulted in a CTD variant incapable of discriminating between membranes of
different composition. Moreover, a L. pneumophila strain producing SidD??loop phenocopied
a L. pneumophila ??sidD strain during growth in mouse macrophages and displayed prolonged
co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting
of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation
during L. pneumophila infection
Internalization and rapid recycling of macrophage Fc receptors tagged with monovalent antireceptor antibody: possible role of a prelysosomal compartment.
The fluorescence protease protection (FPP) assay to determine protein localization and membrane topology
The Mammalian Guanine Nucleotide Exchange Factor mSec12 is Essential for Activation of the Sar1 GTPase Directing Endoplasmic Reticulum Export
Differences in IFNβ secretion upon Rab1 inactivation in cells exposed to distinct innate immune stimuli
A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein
- …