Characterization of Aspergillus nidulansTRAPPs uncovers unprecedented similarities between fungi and metazoans and reveals the modular assembly of TRAPPII.

Mario Pinar,Ernesto Arias-Palomo,Miguel A Peñalva,Vivian De Los Ríos,Herbert N Arst,Alexander Idnurm

doi:10.1371/journal.pgen.1008557

Mario Pinar, Ernesto Arias-Palomo + Show 4 more

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https://doi.org/10.1371/journal.pgen.1008557

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Abstract

TRAnsport Protein Particle complexes (TRAPPs) are ubiquitous regulators of membrane traffic mediating nucleotide exchange on the Golgi regulatory GTPases RAB1 and RAB11. In S. cerevisiae and metazoans TRAPPs consist of two large oligomeric complexes: RAB11-activating TRAPPII and RAB1-activating TRAPPIII. These share a common core TRAPPI hetero-heptamer, absent in metazoans but detected in minor proportions in yeast, likely originating from in vitro-destabilized TRAPPII/III. Despite overall TRAPP conservation, the budding yeast genome has undergone extensive loss of genes, and lacks homologues of some metazoan TRAPP subunits. With nearly twice the total number of genes of S. cerevisiae, another ascomycete Aspergillus nidulans has also been used for studies on TRAPPs. We combined size-fractionation chromatography with single-step purification coupled to mass-spectrometry and negative-stain electron microscopy to establish the relative abundance, composition and architecture of Aspergillus TRAPPs, which consist of TRAPPII and TRAPPIII in a 2:1 proportion, plus a minor amount of TRAPPI. We show that Aspergillus TRAPPIII contains homologues of metazoan TRAPPC11, TRAPPC12 and TRAPPC13 subunits, absent in S. cerevisiae, and establish that these subunits are recruited to the complex by Tca17/TRAPPC2L, which itself binds to the ‘Trs33 side’ of the complex. Thus Aspergillus TRAPPs compositionally resemble mammalian TRAPPs to a greater extent than those in budding yeast. Exploiting the ability of constitutively-active (GEF-independent, due to accelerated GDP release) RAB1* and RAB11* alleles to rescue viability of null mutants lacking essential TRAPP subunits, we establish that the only essential role of TRAPPs is activating RAB1 and RAB11, and genetically classify each essential subunit according to their role(s) in TRAPPII (TRAPPII-specific subunits) or TRAPPII and TRAPPIII (core TRAPP subunits). Constitutively-active RAB mutant combinations allowed examination of TRAPP composition in mutants lacking essential subunits, which led to the discovery of a stable Trs120/Trs130/Trs65/Tca17 TRAPPII-specific subcomplex whose Trs20- and Trs33-dependent assembly onto core TRAPP generates TRAPPII.

Highlights

TRAnsport Protein Particle (TRAPP) complexes regulate various steps of membrane traffic by promoting membrane tethering [1,2,3] and mediating GDP exchange on the RAB GTPases RAB1 and RAB11 [4,5,6,7,8,9,10,11,12]
We show that fungal and metazoan TRAPPs are more similar than previously thought, after identifying three A. nidulans subunits previously believed exclusive to metazoans and demonstrating that TRAPPI is very minor, if it exists at all
Importantly we classified, using a novel genetic approach, essential TRAPP subunits according to their role in activating RAB1 and/or RAB11, which demonstrated that the only indispensable role for TRAPPs is mediating nucleotide exchange on these GTPases and led to the discovery of a stable four-subunit subcomplex that assembles onto the stable seven-subunit core to form the TRAPPII holocomplex

Summary

Introduction

TRAnsport Protein Particle (TRAPP) complexes regulate various steps of membrane traffic by promoting membrane tethering [1,2,3] and mediating GDP exchange on the RAB GTPases RAB1 and RAB11 [4,5,6,7,8,9,10,11,12]. All TRAPPs contain a ‘core hetero-heptamer’, designated TRAPPI in the ascomycete Saccharomyces cerevisiae, consisting of two copies of Bet and one copy each of Trs, Bet, Trs, Trs and Trs (Fig 1A), with Bet3/Bet5/Trs23/Trs being minimally required to activate RABs [6]. Addition of Trs120, Trs130, Trs and Tca to the yeast ‘core hetero-heptamer’ generates TRAPPII [9,13,14], whereas addition of Trs generates TRAPPIII [15] (Fig 1A). These compositional changes are physiologically crucial because they shift the specificity of the GEF between RAB1 (TRAPPI and TRAPPIII) and RAB11 (TRAPPII). Point mutations in RAB11 facilitating spontaneous release of GDP rescue the lethality resulting from ablation of TRAPPII (the RAB11 GEF), but not of core TRAPP/TRAPPI, due to the essential requirement of the latter to activate RAB1 (RabO in A. nidulans [24]; Ypt in S. cerevisiae [25])

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