Kohei Arasaki

Modulation of Rab GTPase function by a protein phosphocholine transferase

The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions.

Legionella pneumophila Promotes Functional Interactions between Plasma Membrane Syntaxins and Sec22b

Biogenesis of a specialized organelle that supports intracellular replication of Legionella pneumophila involves the fusion of secretory vesicles exiting the endoplasmic reticulum (ER) with phagosomes containing this bacterial pathogen. Here, we investigated host plasma membrane SNARE proteins to determine whether they play a role in trafficking of vacuoles containing L. pneumophila. Depletion of plasma membrane syntaxins by RNA interference resulted in delayed acquisition of the resident ER protein calnexin and enhanced retention of Rab1 on phagosomes containing virulent L. pneumophila, suggesting that these SNARE proteins are involved in vacuole biogenesis. Plasma membrane‐localized SNARE proteins syntaxin 2, syntaxin 3, syntaxin 4 and SNAP23 localized to vacuoles containing L. pneumophila. The ER‐localized SNARE protein Sec22b was found to interact with plasma membrane SNAREs on vacuoles containing virulent L. pneumophila, but not on vacuoles containing avirulent mutants of L. pneumophila. The addition of α‐SNAP and N‐ethylmaleimide‐sensitive factor (NSF) to the plasma membrane SNARE complexes formed by virulent L. pneumophila resulted in the dissociation of Sec22b, indicating functional pairing between these SNAREs. Thus, L. pneumophila stimulates the non‐canonical pairing of plasma membrane t‐SNAREs with the v‐SNARE Sec22b to promote fusion of the phagosome with ER‐derived vesicles. The mechanism by which L. pneumophila promotes pairing of plasma membrane syntaxins and Sec22b could provide unique insight into how the secretory vesicles could provide an additional membrane reserve subverted during phagosome maturation.

A Role for the Ancient SNARE Syntaxin 17 in Regulating Mitochondrial Division

Recent evidence suggests that endoplasmic reticulum (ER) tubules mark the sites where the GTPase Drp1 promotes mitochondrial fission via a largely unknown mechanism. Here, we show that the SNARE protein syntaxin 17 (Syn17) is present on raft-like structures of ER-mitochondria contact sites and promotes mitochondrial fission by determining Drp1 localization and activity. The hairpin-like C-terminal hydrophobic domain, including Lys-254, but not the SNARE domain, is important for this regulation. Syn17 also regulates ER Ca(2+) homeostasis and interferes with Rab32-mediated regulation of mitochondrial dynamics. Starvation disrupts the Syn17-Drp1 interaction, thus favoring mitochondrial elongation during autophagy. Because we also demonstrate that Syn17 is an ancient SNARE, our findings suggest that Syn17 is one of the original key regulators for ER-mitochondria contact sites present in the last eukaryotic common ancestor. As such, Syn17 acts as a switch that responds to nutrient conditions and integrates functions for the ER and autophagosomes with mitochondrial dynamics.

Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains

The presence of subdomains in the endoplasmic reticulum (ER) enables this organelle to perform a variety of functions, yet the mechanisms underlying their organization are poorly understood. In the present study, we show that syntaxin 18, a SNAP (soluble NSF attachment protein) receptor localized in the ER, is important for the organization of two ER subdomains, smooth/rough ER membranes and ER exit sites. Knockdown of syntaxin 18 caused a global change in ER membrane architecture, leading to the segregation of the smooth and rough ER. Furthermore, the organization of ER exit sites was markedly changed concomitantly with dispersion of the ER-Golgi intermediate compartment and the Golgi complex. These morphological changes in the ER were substantially recovered by treatment of syntaxin-18-depleted cells with brefeldin A, a reagent that stimulates retrograde membrane flow to the ER. These results suggest that syntaxin 18 has an important role in ER subdomain organization by mediating the fusion of retrograde membrane carriers with the ER membrane.

The Machinery at Endoplasmic Reticulum-Plasma Membrane Contact Sites Contributes to Spatial Regulation of Multiple Legionella Effector Proteins

The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.

Nordihydroguaiaretic Acid Affects Multiple Dynein-Dynactin Functions in Interphase and Mitotic Cells

Nordihydroguaiaretic acid (NDGA), a well known lipoxygenase inhibitor, actually has pleiotropic effects on cells, which include cell proliferation, apoptosis, differentiation, and chemotaxis. We and others have shown previously that this compound causes Golgi disassembly by an unknown mechanism. In this study, we show that, in parallel with Golgi disassembly, NDGA induces the accumulation of the microtubule minus-end-directed motor dynein-dynactin complex at the centrosome, where microtubules minus-ends lie. Concomitant with this accumulation, dynein-dynactin-interacting proteins, such as ZW10 and EB1, were also redistributed to the centrosomal region. In cells where microtubules were depolymerized by nocodazole, NDGA promoted the formation of filaments consisting of dynein-dynactin and its interacting proteins, suggesting that it stimulates the association of these proteins in an ordered, not random, manner. Loss of dynactin function abolished not only NDGA-induced redistribution in intact cells but also filament formation in nocodazole-treated cells. The latter finding implies that dynactin is a key molecule for the association between dynein-dynactin and its interacting proteins. In mitotic cells, NDGA induced robust accumulation of dyneindynactin and its interacting proteins at the spindle poles. These results taken together suggest that NDGA perturbs membrane traffic by affecting the function of the microtubule motor dynein-dynactin complex and its auxiliary proteins. To our knowledge, NDGA is the first case of a reagent that can modulate dynein-dynactin-related processes.

CARTS biogenesis requires VAP-lipid transfer protein complexes functioning at the endoplasmic reticulum-Golgi interface.

Biogenesis of the TGN-derived transport carriers CARTS requires the ER protein VAP and Golgi lipid transfer proteins, ceramide transfer protein and OSBP. Sac1 lipid phosphatase is recruited to a VAP–OSBP complex formed at an ER subdomain closely apposed to the trans-Golgi/TGN. Association–dissociation dynamics of ER–Golgi contacts are important for CARTS formation.

Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17

During infection of macrophages, the pathogenic bacterium Legionella pneumophila secretes effector proteins that induce the conversion of the plasma membrane-derived vacuole into an endoplasmic reticulum (ER)-like replicative vacuole. These ER-like vacuoles are ultimately fused with the ER, where the pathogen replicates. Here we show that the L. pneumophila effector Lpg1137 is a serine protease that targets the mitochondria and their associated membranes. Lpg1137 binds to and cleaves syntaxin 17, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein that is known to participate in the regulation of mitochondrial dynamics through interaction with the mitochondrial fission factor Drp1 in fed cells and in autophagy through interaction with Atg14L and other SNAREs in starved cells. Cleavage of syntaxin 17 inhibits not only autophagy but also staurosporine-induced apoptosis occurring in a Bax, Drp1-dependent manner. Thus, L. pneumophila can shut down ER–mitochondria communication through cleavage of syntaxin 17.

A new role for RINT-1 in SNARE complex assembly at the trans-Golgi network in coordination with the COG complex.

Yeast Tip20, a subunit of the Dsl1 complex, is implicated in Golgi-to–endoplasmic reticulum retrograde transport. Differing from Tip20, its mammalian counterpart, RINT-1, is required for endosome-to–trans-Golgi network transport. RINT-1 in coordination with the COG complex regulates SNARE complex assembly at the trans-Golgi network.

Contribution of the long form of syntaxin 5 to the organization of the endoplasmic reticulum.

Summary The SNARE protein syntaxin 5 exists as long (42 kDa) and short (35 kDa) isoforms. The short form is principally localized in the Golgi complex, whereas the long form resides not only in the Golgi but also in the endoplasmic reticulum (ER). Although the Golgi-localized short form has been extensively investigated, little is known about the long form. In the present study, we demonstrate that the long form of syntaxin 5 functions to shape the ER. We found that overexpression of the long form of syntaxin 5 induces rearrangement and co-alignment of the ER membrane with microtubules, the pattern of which is quite similar to that observed in cells overexpressing CLIMP-63, a linker between the ER membrane and microtubules. The ability of syntaxin 5 to induce ER-microtubule rearrangement is not related to its SNARE function, but correlates with its binding affinities for CLIMP-63, and CLIMP-63 is essential for the induction of this rearrangement. Microtubule co-sedimentation assays demonstrated that the long form of syntaxin 5 has a substantial microtubule-binding activity. These results suggest that the long form of syntaxin 5 contributes to the regulation of ER structure by interacting with both CLIMP-63 and microtubules. Indeed, depletion of syntaxin 5 caused the spreading of the ER to the cell periphery, similar to the phenotype observed in cells treated with the microtubule-depolymerizing reagent nocodazole. Our results disclose a previously undescribed function of the long form of syntaxin 5 that is not related to its function as a SNARE.