Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) cause late-onset Parkinson's disease indistinguishable from idiopathic disease. The mechanisms whereby missense alterations in the LRRK2 gene initiate neurodegeneration remain unknown. Here, we demonstrate that seven of 10 suspected familial-linked mutations result in increased kinase activity. Functional and disease-associated mutations in conserved residues reveal the critical link between intrinsic guanosine triphosphatase (GTPase) activity and downstream kinase activity. LRRK2 kinase activity requires GTPase activity, whereas GTPase activity functions independently of kinase activity. Both LRRK2 kinase and GTPase activity are required for neurotoxicity and potentiate peroxide-induced cell death, although LRRK2 does not function as a canonical MAP-kinase-kinase-kinase. These results suggest a link between LRRK2 kinase activity and pathogenic mechanisms relating to neurodegeneration, further supporting a gain-of-function role for LRRK2 mutations.

Current models suggest that TRAPP tethering complexes exist in two forms. Whereas the seven-subunit TRAPPI complex mediates ER-to-Golgi transport, TRAPPII contains three additional subunits (Trs65, Trs120 and Trs130) and is required for distinct tethering events at Golgi membranes. It is not clear how TRAPPII assembly is regulated. Here, we show that Tca17 is a fourth TRAPPII-specific component, and that Trs65 and Tca17 interact with distinct domains of Trs130 and make different contributions to complex assembly. Whereas Tca17 promotes the stable association of TRAPPII-specific subunits with the core complex, Trs65 stabilizes TRAPPII in an oligomeric form. We show that Trs85, which was previously reported to be a subunit of both TRAPPI and TRAPPII, is not associated with the TRAPPII complex in yeast. However, we find that proteins related to Trs85, Trs65 and Tca17 are part of the same TRAPP complex in mammalian cells. These findings have implications for models of TRAPP complex formation and suggest that TRAPP complexes may be organized differently in yeast and mammals. The specificity of vesicle transport is a highly regulated process. Tethering complexes provide the initial recognition event that links a particular vesicle with its target membrane, while the subsequent pairing of cognate SNARE proteins on both membranes drives the fusion of lipid bilayers (1,2). Many tethers are recruited to the correct membrane by binding to activated Rab GTPases and SNARE regulatory domains, and recognize components of the vesicle coat. Some tethers additionally act as GTP exchange factors (GEFs) to promote Rab activation.Tethers take the form of extended coiled-coil proteins or large multisubunit complexes (2). Yeast TRAPPI, one of the best-characterized multisubunit tethering complexes, is required for ER-Golgi transport and is a GEF for the Rab protein Ypt1 (3-6). Current models suggest that TRAPPI contains seven subunits (Bet3, Bet5, Trs31, Trs23, Trs33, Trs20, Trs85), four of which are needed for GEF activity (3,7). Structural analyses demonstrate that Trs23, Bet3 and Bet5 form the interface that binds Ypt1, while Trs31 may stabilize the interface (8). Bet3, which is present in two copies, additionally binds the Sec23 subunit of the coat protein II (COPII) coat, thus linking the vesicle coat to Rab activation (9).A second form of the TRAPP complex, TRAPPII, participates in intra-Golgi and endosome-Golgi transport (10,11). Yeast TRAPPII is formed by the addition of three new subunits -Trs120, Trs130, Trs65 -to TRAPPI (6). TRAPPII does not bind COPII vesicles, but instead recognizes a component of the COPI coat (10). While some studies suggest that conversion of TRAPPI to TRAPPII is also accompanied by a switch in GEF activity (11), others find both forms of TRAPP are GEFs for Ypt1 (8). Nevertheless, it is generally agreed that TRAPPII -directly or indirectly -functions upstream of Ypt31/Ypt32 activation in Golgi trafficking.The function of TRAPPII appears to be conserved in mammalian cells, where it binds COPI and regula...

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Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N-and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.