Understanding the molecular basis of Rab-GEF activity and specificity of the mammalian TRAPP complexes




Harris, Noah James

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Rab GTPases are among the most important families of proteins involved in regulating the trafficking and delivery of cellular cargo to their proper locations. The multi-subunit Transport Protein Particle complexes, TRAPPII and TRAPPIII, activate Rabs by catalyzing GDP/GTP nucleotide exchange. These two distinct complexes share seven subunits, yet they differ in their complex-specific subunits. Even though the TRAPP complexes are similar in subunit composition, they can activate different Rabs and therefore regulate distinct trafficking pathways. Intriguingly, the mechanism underlying the specificity of TRAPPII/III for different Rabs is poorly understood. This thesis is centered around understanding the molecular basis of TRAPP-Rab specificity in the mammalian TRAPPII/III complexes. To address this, we used a combination of different powerful techniques including biochemical nucleotide exchange assays, Hydrogen Deuterium eXchange Mass Spectrometry (HDX-MS), and electron microscopy to understand this specificity both in solution and on membranes. Biochemical assays against 20 different Rab GTPases revealed that TRAPPIII only has activity on Rab1 and Rab43. HDX-MS experiments comparing the TRAPPII and TRAPPIII complexes showed that there are extensive differences existing near the Rab binding site, thus highlighting the critical role that the complex specific subunits serve in modifying either complex’s Rab binding interface. TRAPPII and TRAPPIII showed increased activity in the presence of lipid membranes and HDX-MS revealed large dynamic changes occurring in both TRAPP complexes in the presence of membranes. Altogether, the work summarized in this thesis provides novel insight into the unique functions of the two TRAPP complexes and it reveals how the complex specific subunits can rearrange the Rab binding site which leads to a possible mechanism for Rab specificity.



Rab GTPases, Guanine nucleotide exchange factors, TRAPP, Membrane trafficking