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1.
bioRxiv ; 2024 Jul 13.
Article in English | MEDLINE | ID: mdl-39026773

ABSTRACT

Multicellularity was accompanied by the emergence of new classes of cell surface and secreted proteins. The nematode C. elegans is a favorable model to study cell surface interactomes, given its well-defined and stereotyped cell types and intercellular contacts. Here we report our C. elegans extracellular interactome dataset, the largest yet for an invertebrate. Most of these interactions were unknown, despite recent datasets for flies and humans, as our collection contains a larger selection of protein families. We uncover new interactions for all four major axon guidance pathways, including ectodomain interactions between three of the pathways. We demonstrate that a protein family known to maintain axon locations are secreted receptors for insulins. We reveal novel interactions of cystine-knot proteins with putative signaling receptors, which may extend the study of neurotrophins and growth-factor-mediated functions to nematodes. Finally, our dataset provides insights into human disease mechanisms and how extracellular interactions may help establish connectomes.

2.
J Biol Chem ; 299(12): 105441, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37949220

ABSTRACT

MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca2+-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dynein-dynactin. The mechanism by which MIRO interacts with TRAK is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca2+ and/or GTP binding. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1's EF-hands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394 to 431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1's EF-hands bind Ca2+ with dissociation constants (KD) of 3.9 µM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca2+ whereas the other EF-hand binds Ca2+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca2+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.


Subject(s)
Kinesins , Mitochondria , Humans , GTP Phosphohydrolases/metabolism , Guanosine Triphosphate/metabolism , Kinesins/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , rho GTP-Binding Proteins/metabolism
3.
J Cell Sci ; 136(5)2023 03 01.
Article in English | MEDLINE | ID: mdl-36744380

ABSTRACT

Mitochondrial homeostasis requires a dynamic balance of fission and fusion. The actin cytoskeleton promotes fission, and we found that the mitochondrially localized myosin, myosin 19 (Myo19), is integral to this process. Myo19 knockdown induced mitochondrial elongation, whereas Myo19 overexpression induced fragmentation. This mitochondrial fragmentation was blocked by a Myo19 mutation predicted to inhibit ATPase activity and strong actin binding but not by mutations predicted to affect the working stroke of the motor that preserve ATPase activity. Super-resolution imaging indicated a dispersed localization of Myo19 on mitochondria, which we found to be dependent on metaxins. These observations suggest that Myo19 acts as a dynamic actin-binding tether that facilitates mitochondrial fragmentation. Myo19-driven fragmentation was blocked by depletion of either the CAAX splice variant of the endoplasmic reticulum (ER)-anchored formin INF2 or the mitochondrially localized F-actin nucleator Spire1C (a splice variant of Spire1), which together polymerize actin at sites of mitochondria-ER contact for fission. These observations imply that Myo19 promotes fission by stabilizing mitochondria-ER contacts; we used a split-luciferase system to demonstrate a reduction in these contacts following Myo19 depletion. Our data support a model in which Myo19 tethers mitochondria to ER-associated actin to promote mitochondrial fission.


Subject(s)
Actins , Mitochondrial Dynamics , Actins/metabolism , Myosins/metabolism , Mitochondria/metabolism , Endoplasmic Reticulum/metabolism
4.
Proc Natl Acad Sci U S A ; 119(29): e2119695119, 2022 07 19.
Article in English | MEDLINE | ID: mdl-35858355

ABSTRACT

Human glucose-6-phosphate dehydrogenase (G6PD) is the main cellular source of NADPH, and thus plays a key role in maintaining reduced glutathione to protect cells from oxidative stress disorders such as hemolytic anemia. G6PD is a multimeric enzyme that uses the cofactors ß-D-glucose 6-phosphate (G6P) and "catalytic" NADP+ (NADP+c), as well as a "structural" NADP+ (NADP+s) located ∼25 Å from the active site, to generate NADPH. While X-ray crystallographic and biochemical studies have revealed a role for NADP+s in maintaining the catalytic activity by stabilizing the multimeric G6PD conformation, other potential roles for NADP+s have not been evaluated. Here, we determined the high resolution cryo-electron microscopy structures of human wild-type G6PD in the absence of bound ligands and a catalytic G6PD-D200N mutant bound to NADP+c and NADP+s in the absence or presence of G6P. A comparison of these structures, together with previously reported structures, reveals that the unliganded human G6PD forms a mixture of dimers and tetramers with similar overall folds, and binding of NADP+s induces a structural ordering of a C-terminal extension region and allosterically regulates G6P binding and catalysis. These studies have implications for understanding G6PD deficiencies and for therapy of G6PD-mediated disorders.


Subject(s)
Glucosephosphate Dehydrogenase , NADP , Catalytic Domain/genetics , Cryoelectron Microscopy , Glucosephosphate Dehydrogenase/chemistry , Glucosephosphate Dehydrogenase/genetics , Glucosephosphate Dehydrogenase Deficiency/drug therapy , Glucosephosphate Dehydrogenase Deficiency/enzymology , Humans , Mutation , NADP/chemistry , Protein Multimerization
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