The substrate specificity of the human TRAPPII complex’s Rab-guanine nucleotide exchange factor activity

Jenkins, Meredith L.; Harris, Noah J; Dalwadi, Udit; Fleming, Kaelin D; Ziemianowicz, Daniel S.; Rafiei, Atefeh; Martin, Emily Medlin; Schriemer, David C.; Yip, Calvin K.; Burke, John E.

doi:10.1038/s42003-020-01459-2

Cited by 17 publications

(35 citation statements)

References 85 publications

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“…Dimerization may be important for enforcing the orientation of yeast TRAPPII on the membrane surface, so it is an open question whether the metazoan TRAPPII complex also employs steric gating to counterselect against Rab1. Nevertheless, comparison of our findings to published low-resolution structures, crosslinking data, and structural predictions of TRAPPII from other organisms (26,27,29) (figs. S5 and S6), indicates the overall organization of the TRAPPII monomer is conserved across eukaryotes.…”

Section: Discussionmentioning

confidence: 53%

“…In the yeast TRAPPII complex, Trs65 creates an extensive interface to dimerize the complex. Metazoan TRAPPII complexes lack a homolog of the Trs65 subunit and are correspondingly monomeric ( 26, 27, 30, 32, 40 ). Dimerization may be important for enforcing the orientation of yeast TRAPPII on the membrane surface, so it is an open question whether the metazoan TRAPPII complex also employs steric gating to counterselect against Rab1.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, cryoEM structures of the intact yeast and fly TRAPPIII complexes were reported (25,26); these studies elucidated the architecture of TRAPPIII and determined how it binds membranes during the Rab activation reaction. Low resolution structures of TRAPPII from multiple organisms have also been determined (26)(27)(28)(29)), yet the central question of how the same active site can activate different Rab substrates in different contexts remains unresolved. It is not known why TRAPPIII does not activate Rab11, how TRAPPII is able to activate Rab11, or how TRAPPII counterselects against Rab1 by steric gating.…”

Section: Introductionmentioning

confidence: 99%

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Structure of a TRAPPII-Rab11 activation intermediate reveals GTPase substrate selection mechanisms

Bagde

Fromme

2021

Preprint

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Rab1 and Rab11 are essential regulators of the eukaryotic secretory and endocytic recycling pathways. The TRAPP complexes activate these GTPases via nucleotide exchange using a shared set of core subunits. The basal specificity of the TRAPP core is towards Rab1, yet the TRAPPII complex is specific for Rab11. A steric gating mechanism has been proposed to explain TRAPPII counterselection against Rab1. Here we present cryoEM structures of the 22-subunit TRAPPII complex from budding yeast, including a TRAPPII-Rab11 nucleotide exchange intermediate. The Trs130 subunit provides a ″leg″ that positions the active site distal to the membrane surface, and this leg is required for steric gating. The related TRAPPIII complex is unable to activate Rab11 due to a repulsive interaction, which TRAPPII surmounts using the Trs120 subunit as a ″lid″ to enclose the active site. TRAPPII also adopts an open conformation enabling Rab11 to access and exit from the active site chamber.

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Section: Discussionmentioning

confidence: 53%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structure of a TRAPPII-Rab11 activation intermediate reveals GTPase substrate selection mechanisms

Bagde

Fromme

2021

Preprint

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“…and Rab43 [10]. The TRAPPIII complex is a well validated GEF for Rab1a [11,14,15,35], however, the substrate specificity towards other Rab GTPases for mammalian TRAPPIII is unknown.…”

Section: Discussionmentioning

confidence: 99%

“…TRAPPII complexe and Rabs were purified as described previously [10]. TRAPP cell pellets were lysed by sonication for 1. concentrator and size exclusion chromatography (SEC) was performed using a Superose 6 increase 10/300 column equilibrated in SEC Buffer.…”

Section: Protein Purificationmentioning

confidence: 99%

Biochemical insight into novel Rab-GEF activity of the mammalian TRAPPIII complex

Harris

Jenkins

Dalwadi

et al. 2021

Preprint

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Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that the TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.

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The TRAPP complexes: oligomeric exchange factors that activate the small GTPases Rab1 and Rab11

Galindo

Munro

2022

FEBS Letters

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The Transport Protein Particle (TRAPP) complexes are highly conserved multisubunit complexes that act as nucleotide exchange factors (GEFs) for Rab GTPases. They act in both protein secretion and autophagy and have also been proposed to have a role in other processes such as cytokinesis and ciliogenesis. There are two TRAPP complexes in metazoans: TRAPPII, which activates Rab11; and TRAPPIII, which activates Rab1. Both complexes share a core of small subunits that form the active site for the exchange of GDP for GTP. In addition, each TRAPP complex has distinct large subunits that determine the specificity of each complex towards its substrate Rab and are essential for activity in vivo. Crystal structures have revealed the organisation of the TRAPP core and the mechanism of Rab1 activation, whilst recent cryo‐EM structures have unveiled the arrangement of the specific subunits around the core to form each complex. Combining these findings with functional experiments has allowed the proposal of mechanisms for how the specificity of each complex towards their cognate Rab is determined and for the arrangement of these large complexes on the membrane.

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The substrate specificity of the human TRAPPII complex’s Rab-guanine nucleotide exchange factor activity

Cited by 17 publications

References 85 publications

Structure of a TRAPPII-Rab11 activation intermediate reveals GTPase substrate selection mechanisms

Structure of a TRAPPII-Rab11 activation intermediate reveals GTPase substrate selection mechanisms

Biochemical insight into novel Rab-GEF activity of the mammalian TRAPPIII complex

The TRAPP complexes: oligomeric exchange factors that activate the small GTPases Rab1 and Rab11

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