Biochemical Insight into Novel Rab-GEF Activity of the Mammalian TRAPPIII Complex
| Autor: | Sung-Eun Nam, Meredith L. Jenkins, Matthew A.H. Parson, John E. Burke, Kaelin D. Fleming, Udit Dalwadi, Calvin K. Yip, Noah J Harris |
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| Rok vydání: | 2021 |
| Předmět: |
Protein Conformation
Vesicular Transport Proteins GTPase 03 medical and health sciences 0302 clinical medicine Structural Biology Animals Guanine Nucleotide Exchange Factors Humans Binding site Molecular Biology 030304 developmental biology Mammals 0303 health sciences Binding Sites biology Chemistry Protein dynamics fungi 030302 biochemistry & molecular biology Cell Membrane Active site RAB1 Cell biology Protein Transport rab GTP-Binding Proteins biology.protein Hydrogen–deuterium exchange Guanine nucleotide exchange factor Rab 030217 neurology & neurosurgery |
| Zdroj: | Journal of molecular biology. 433(18) |
| ISSN: | 1089-8638 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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