RESUMEN
The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Complejos Multiproteicos/metabolismo , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Proteínas Relacionadas con la Autofagia/química , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Células HEK293 , Humanos , Espectrometría de Masas , Unión Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Rab proteins are a large family of GTP-binding proteins that regulate cellular membrane traffic and organelle identity. Rab proteins cycle between association with membranes and binding to RabGDI. Bound on membranes, each Rab has a very specific cellular location and it is this remarkable degree of specificity with which Rab GTPases recognize distinct subsets of intracellular membranes that forms the basis of their ability to act as key cellular regulators, determining the recruitment of downstream effectors to the correct membrane at the correct time. The molecular mechanisms controlling Rab localization remain poorly understood. Here, we present a fluorescence-based assay to investigate Rab GTPase membrane extraction and delivery by RabGDI. Using EGFP-Rab fusion proteins the amount of Rab:GDI complex obtained by GDI extraction of Rab proteins from HEK293 membranes could be determined, enabling control of complex concentration. Subsequent partitioning of the Rab GTPases into vesicles made up of artificial binary lipid mixtures showed for the first time, that the composition of the target membrane plays a key role in the localization of Rab proteins by sensing the stored curvature elastic energy in the membrane.
Asunto(s)
Membrana Celular/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Membrana Celular/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Lípidos de la Membrana/genética , Unión Proteica/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab5/genéticaRESUMEN
3-(3-Pyridyl)-2-hydroxy-2-phosphonopropanoic acid (3-PEHPC, 1) is a phosphonocarboxylate (PC) analogue of 2-(3-pyridyl)-1-hydroxyethylidenebis(phosphonic acid) (risedronic acid, 2), an osteoporosis drug that decreases bone resorption by inhibiting farnesyl pyrophosphate synthase (FPPS) in osteoclasts, preventing protein prenylation. 1 has lower bone affinity than 2 and weakly inhibits Rab geranylgeranyl transferase (RGGT), selectively preventing prenylation of Rab GTPases. We report here the synthesis and biological studies of 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC, 3), the PC analogue of minodronic acid 4. Like 1, 3 selectively inhibited Rab11 vs. Rap 1A prenylation in J774 cells, and decreased cell viability, but was 33-60x more active in these assays. After resolving 3 by chiral HPLC (>98% ee), we found that (+)-3-E1 was much more potent than (-)-3-E2 in an isolated RGGT inhibition assay, approximately 17x more potent (LED 3 microM) than (-)-3-E2 in inhibiting Rab prenylation in J774 cells and >26x more active in the cell viability assay. The enantiomers of 1 exhibited a 4-fold or smaller potency difference in the RGGT and prenylation inhibition assays.
