A working model summarizes our principle findings of how DrrA directs the fusion of ER-derived vesicles with the PM-derived organelle containing Legionella
and underscores new open quesitons (). Stage 1 depicts interactions mediated by the GEF domain and the PI4P-binding domain of DrrA. Our data indicate that the DrrA carboxy-terminal region interacts directly with PM-localized syntaxins (), providing an additional function to a domain that was previously shown to have a PI4P binding activity important for localization of DrrA to PM-derived organelles (Brombacher et al., 2009
; Schoebel et al., 2010
; Zhu et al., 2010
). The interaction with Stx3 and the full-length DrrA protein was observed upon infection of Legionella
(), and could be enhanced in vitro
by the binding of inactive GDP-bound Rab1 to the GEF domain (). The dynamics of Rab1 activation by the GEF domain in DrrA leads to the accumulation of Rab1-GTP on the PM-derived compartment and interactions mediated by the PI4P-binding domain in DrrA would facilitate the localization of syntaxins to this region of the membrane. Thus, these activities of DrrA serve to localize host factors that participate in the tethering and fusion of ER-derived vesicles with the PM-derived organelle.
Stage 2 depicts the Rab1-dependent tethering of ER-derived vesicles to the PM-derived compartment. This stage is supported by data showing in permeabilized cells that the tethering of ER-derived vesicles to the PM required DrrA with a functional GEF domain, Rab1, and GTP (Figures S3-S5
and ). A role for SNARE proteins in the tethering stage was not apparent, as silencing of either Sec22b or PM syntaxins did not reduce vesicle tethering stimulated by DrrA. Given that Rab-mediated vesicle tethering in cells usually involves a protein that binds to a GTP-bound Rab protein on the acceptor membrane and associates with factors on the membrane of the donor vesicle, an unidentified tethering factor that binds to active Rab1 and interacts with the ER-derived vesicle is depicted in this model. This protein could be a factor that normally associates with early secretory vesicles and binds active Rab1 on Golgi or pre-Golgi membranes. Alternatively, this tethering factor could be a protein that is not typically associated with early secretory vesicles. Future studies are needed to determine the identity of this putative tethering factor and other host factors that may participate in this Rab1-mediated tethering reaction.
Stage 3 depicts the functional association of Sec22b on the ER-derived vesicles with the PM Stx3 protein. Our data indicate that formation of this complex can be stimulated by DrrA-mediated activation of Rab1 (). Interestingly, homologues of these SNARE proteins from Saccharomyces cerevisiae
promote membrane fusion in vitro
when incorporated into synthetic vesicles, however, it was argued that fusion of ER-derived vesicles with the PM would be restricted topologically by cellular mechanisms that regulate vesicle tethering in vivo
(McNew et al., 2000
). Consistent with this hypothesis, our model predicts that the tethering reaction mediated by activated Rab1 at the PM is sufficient to overcome this topological restriction and promote functional interactions between Sec22b and PM syntaxins. Thus, the primary role of DrrA in promoting the tethering of ER-derived vesicles with the PM is to overcome this topological restriction by activating Rab1 on this organelle.
Stage 4 depicts the fusion of ER-derived vesicles with the PM-derived organelle. Independent indicators of this fusion reaction are provided by data showing the DrrA-mediated release of the soluble Luciferase-KDEL protein contained in the lumen of ER-derived vesicles and the delivery of the transmembrane protein ts045-VSVG-GFP from the limiting membrane of ER-derived vesicles to the surface of cells (). Fusion of ER-derived vesicles with the PM resulting from DrrA-mediated activation of Rab1 was blocked when PM syntaxins were reduced on the acceptor compartment or when Sec22b was reduced on the donor compartments, demonstrating fusion involves a topological requirement for these SNAREs on distinct organelles. SNAP23 is depicted as also being involved in this fusion process based on data showing fusion of synthetic liposomes with a t-SNARE complex consisting of the SNAP23 homologue Sec9p and the PM syntaxin homologue Sso1p with liposomes that display the v-SNARE Sec22p (McNew et al., 2000
Importantly, the observation that DrrA was sufficient to promote the tethering and fusion of ER-derived vesicles with the PM indicates that no additional bacterial factors are required to promote this reaction. In the context of Legionella
infection, however, other effector proteins are predicted to enhance the efficiency of this pathway. Notably, the Legionella
effector LidA binds to Rab1, and immobilization of LidA and DrrA to paramagnetic beads is sufficient to stimulate the tethering of ER-derived vesicles to beads by a Rab1-dependent mechanism (Machner and Isberg, 2006
). Thus, LidA should enhance DrrA-mediated fusion of ER-derived vesicles with the PM-derived organelle. Additionally, the observation that Dot/Icm-dependent Sec22b and Stx3 interactions are detected during infection of host cells by a ΔdrrA
mutant (Arasaki and Roy, 2010
) indicate that there should be Legionella
effectors that promote tethering of ER-derived vesicles to the LCV membrane by a process that does not require DrrA function. Possibilities include effectors that mimic active Rab1 or mimic the tethering factors Rab1 recruits to the LCV. Tethering mediated by these effectors would then promote the engagement of PM syntaxins and Sec22b, which would promote fusion of ER-derived vesicles with the LCV by a pathway that is independent of DrrA.
Lastly, these data may provide insight into cellular process that could direct the fusion of ER-derived vesicles with phagosome membranes during endocytic maturation. There are several independent studies that suggest the existence of an endogenous cellular pathway in phagocytic cells, such as macrophages, that enables ER-derived vesicles to fuse with phagosomes (Becker et al., 2005
; Gagnon et al., 2002
; Hatsuzawa et al., 2006
). Immunological studies have indicated that fusion of ER with phagosomes is important for the delivery components that allow antigens in the lumen of phagosomes to be transported across a vacuole membrane and presented on MHC class I molecules (Ackerman et al., 2006
; Guermonprez et al., 2003
; Houde et al., 2003
). However, it is difficult to detect fusion of ER-derived membranes with phagosomes by fluorescence microscopy (Touret et al., 2005
) or by quantitative mass spectrometry (Rogers and Foster, 2007
), suggesting if fusion of ER-derived membranes with phagosomes represents an endogenous transport pathway in cells, this process must be tightly regulated. Our data suggest that activation of Rab1 on the phagosome membrane would be sufficient to promote the recruitment of ER-derived vesicles, and fusion of these vesicles with the phagosomes would occur upon formation of the SNARE complex containing Sec22b and PM syntaxins. Thus, it will be interesting to know whether phagocytic cells produce a GEF that functions analogously to DrrA to regulate the recruitment and fusion of ER-derived vesicles with phagosomes, and if Sec22b and PM syntaxins are involved.