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

Abstract

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.

Molecular basis for the recruitment of the Rab effector protein WDR44 by the GTPase Rab11

“…The full-length Rab genes were used as previously described ( 36 , 37 ). The WDR44 gene was purchased from the DNASU plasmid repository (HsCD00296048).…”

Deep proteomics identifies shared molecular pathway alterations in synapses of schizophrenia and bipolar disorder patients and mouse model

“…It is known that COPII as well as TRAPPIII complex proteins, which are both elevated in SCZ/BP synapses (Fig. 4G), can promote autophagy during nutrient stress (Tan et al, 2013; Jeong et al, 2018; Harris et al, 2021). These findings suggest that autophagy may be activated at SCZ and BP synapses.…”

Functional Characterization of a missense mutation in the TRAPPC12 gene presenting with progressive encephalopathy with brain atrophy and spasticity phenotype without microcephaly and epilepsy

“…The discovery of speci c subunits of the TRAPP complexes contributed to the molecular processes between the ER-ERGIC-Golgi apparatus (21). Remarkably, two different complexes exist in metazoans and humans, TRAPP II and TRAPP III (9). One study about the cryo-EM structure of the metazoan TRAPP III demonstrated that two arms and one core of the complex exist.…”

“…One of the main functions of these complexes is the tra cking interaction between the endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC), and Golgi apparatus (3,4,7). Twenty years of subsequent research studies showed TRAPP complexes mediate the interaction between ER-exit-sites (COPI and COPII vesicles) through the Golgi apparatus (8) and show guanine nucleotide exchange factor (GEF) activity (9). This recent study showed that various conformational changes in the TRAPP complex have a vigorous GEF activity, including Rab1 and Rab11 interacting with TRAPP II, and particularly TRAPP III has a distinct speci city to Rab1 (9).…”

“…Twenty years of subsequent research studies showed TRAPP complexes mediate the interaction between ER-exit-sites (COPI and COPII vesicles) through the Golgi apparatus (8) and show guanine nucleotide exchange factor (GEF) activity (9). This recent study showed that various conformational changes in the TRAPP complex have a vigorous GEF activity, including Rab1 and Rab11 interacting with TRAPP II, and particularly TRAPP III has a distinct speci city to Rab1 (9). In addition, TRAPP III complexes have a role in kinetochore stability, mitotic transformation, and autophagy (10)(11)(12).…”