title: TRAPPII regulates exocytic Golgi exit by mediating nucleotide exchange on the Ypt31 ortholog RabERAB11.
abstract: The oligomeric complex transport protein particle I (TRAPPI) mediates nucleotide exchange on the RAB GTPase RAB1/Ypt1. TRAPPII is composed of TRAPPI plus three additional subunits, Trs120, Trs130, and Trs65. Unclear is whether TRAPPII mediates nucleotide exchange on RAB1/Ypt1, RAB11/Ypt31, or both. In Aspergillus nidulans, RabO(RAB1) resides in the Golgi, RabE(RAB11) localizes to exocytic post-Golgi carriers undergoing transport to the apex, and hypA encodes Trs120. RabE(RAB11), but not RabO(RAB1), immunoprecipitates contain Trs120/Trs130/Trs65, demonstrating specific association of TRAPPII with RabE(RAB11) in vivo. hypA1(ts) rapidly shifts RabE(RAB11), but not RabO(RAB1), to the cytosol, consistent with HypA(Trs120) being specifically required for RabE(RAB11) activation. Missense mutations rescuing hypA1(ts) at 42 °C mapped to rabE, affecting seven residues. Substitutions in six, of which four resulted in 7- to 36-fold accelerated GDP release, rescued lethality associated to TRAPPII deficiency, whereas equivalent substitutions in RabO(RAB1) did not, establishing that the essential role of TRAPPII is facilitating RabE(RAB11) nucleotide exchange. In vitro, TRAPPII purified with HypA(Trs120)-S-tag accelerates nucleotide exchange on RabE(RAB11) and, paradoxically, to a lesser yet substantial extent, on RabO(RAB1). Evidence obtained by exploiting hypA1-mediated destabilization of HypA(Trs120)/HypC(Trs130)/Trs65 assembly onto the TRAPPI core indicates that these subunits sculpt a second RAB binding site on TRAPP apparently independent from that for RabO(RAB1), which would explain TRAPPII in vitro activity on two RABs. Using A. nidulans in vivo microscopy, we show that HypA(Trs120) colocalizes with RabE(RAB11), arriving at late Golgi cisternae as they dissipate into exocytic carriers. Thus, TRAPPII marks, and possibly determines, the Golgi-to-post-Golgi transition.
authors.affiliation: Departments of Cellular and Molecular Biology, and.
authors.forename: Mario
authors.initials: M
authors.lastname: Pinar
authors.affiliation: Departments of Cellular and Molecular Biology, and Section of Microbiology, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Herbert N
authors.initials: HN
authors.lastname: Arst
authors.affiliation: Departments of Cellular and Molecular Biology, and.
authors.forename: Areti
authors.initials: A
authors.lastname: Pantazopoulou
authors.affiliation: Departments of Cellular and Molecular Biology, and.
authors.forename: Víctor G
authors.initials: VG
authors.lastname: Tagua
authors.affiliation: Departments of Cellular and Molecular Biology, and.
authors.forename: Vivian
authors.initials: V
authors.lastname: de los Ríos
authors.affiliation: Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain; and.
authors.forename: Javier
authors.identifier: http://orcid.org/0000-0002-2932-0142
authors.initials: J
authors.lastname: Rodríguez-Salarichs
authors.affiliation: Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain; and.
authors.forename: J Fernando
authors.initials: JF
authors.lastname: Díaz
authors.affiliation: Departments of Cellular and Molecular Biology, and penalva@cib.csic.es.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Peñalva
mesh_terms: D001233:Aspergillus nidulans / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005089:Exocytosis; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D020125:Mutation, Missense; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
chemical_list: D005656:Fungal Proteins; D005982:Glutathione Transferase; D049452:Green Fluorescent Proteins; D006153:Guanosine Diphosphate; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C406327:transport protein particle, TRAPP; C087905:YPT31 protein, S cerevisiae; C060755:YPT1 protein, S cerevisiae
keywords: Aspergillus; GEF; GTPase; Golgi; post-Golgi carriers
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