Characterization of the role of TRAPPC2/Trs20p-related and associated proteins in membrane traffic

In the process of intracellular trafficking, fidelity of delivering proteins and lipids across the secretory pathway is of critical importance. Any failure in this highly regulated event could have severe consequences to the cell. Various human diseases arise from mutations affecting membrane trafficking. In this regard, vesicle tethering complexes serve as key factors for the maintenance of cellular function. The transport protein particle (TRAPP) is one such factor which provides specificity in delivering proteins and lipids. TRAPP is found in three related complexes sharing core subunits, each governing different transport steps. TRAPPC2, a mammalian ortholog of yeast TRS20, is an essential gene that codes for a protein that exists in all forms of the TRAPP complexes. My research has focused on elucidating the cellular function of TRAPPC2 and proteins that associate with it. Substitution of an aspartic acid residue at position 47 of TRAPPC2 to tyrosine has been shown to cause a skeletal disorder known as spondyloepiphyseal dysplasia tarda (SEDT), a disorder which is believed to be due to a defect in collagen secretion. In Chapter 2 I demonstrate that aspartic acid residue 47 is absolutely invariant across taxa suggesting that this amino acid plays an important role in the function of the TRAPP complex. Even though TRAPPC2 is ubiquitously expressed the SEDT phenotype is manifested in only in specific tissue. Thus, we rationalised a search for homologs of TRAPPC2/Trs20p, hoping to provide an answer to the tissue-specificity of SEDT. We identified two novel proteins; TRAPPC2L and its yeast counterpart Tca17p. The position for the novel TRAPPC2L protein is postulated to be opposite to the region where TRAPPC2/Trs20p incorporates into the TRAPP complex. In Chapter 3 I demonstrate a direct interaction between the TRAPP complex and the SNARE fusion machinery. This binding is lost in the pathogenic TRAPPC2D47Y mutant. Subsequently, we revealed that an SEDT-analogous mutation in Trs20p (trs20D46Y) resulted in deficiency in autophagy rather than defects in endoplasmic reticulum to Golgi trafficking. Chapter 4 describes the discovery of the association between TRAPP and the tethering factor p115. By using the TRAPPC2D47Y mutant I showed that p115 could not efficiently dissociate from membranes, thereby showing that a TRAPP-p115 interaction is critical for p115 membrane recognition. Furthermore, I provide evidence that TRAPP associates with p115 and SNAREs in a Brefeldin A-resistant manner. I propose placing this association at the ER-Golgi intermediate compartment (ERGIC) membranes, a compartment that lacks in lower eukaryotic cells, at the very early stage of the secretory pathway. Previous work by our laboratory found several novel mammalian TRAPP components including TRAPPC11. Chapter 5 discusses our discoveries into the function of TRAPPC11, a TRAPPC2 protein partner. The genetic component of this work was conducted by our collaborators from Alberta who used homozygosity mapping in combination with exome sequencing in two siblings from a Hutterite family. They found that the candidate gene mutation affects the foie gras domain of TRAPPC11 in these brothers. The deletion mutation accounts for the array of phenotypes including myopathy, ataxia, and intellectual disability (ID) that is observed in these patients. I demonstrated that this mutation disrupts TRAPPC11 binding to multiple TRAPP subunits including TRAPPC2 and compromises the integrity of the Golgi apparatus. I also showed that this mutation causes a dramatic delay in trafficking from the Golgi to the plasma membrane. Moreover, this mutation dramatically affects the localization of lysosomal membrane glycoprotein 1 (LAMP1). This is the first study to investigate the function of the foie gras domain of TRAPPC11 in humans. Finally, in Chapter 6 I discuss the implications of all of the studies performed in the preceding chapters and provide a working model for the function of TRAPPC11 in membrane traffic.