TRAPPC2

Abstract: Mutations in the trafficking protein particle complex C2 protein (TRAPPC2), a mammalian ortholog of yeast Trs20p and a component of the trafficking protein particle (TRAPP) vesicle tethering complex, have been linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT). Intriguingly, the X-linked TRAPPC2 is just one of a complement of Trs20-related genes in humans. Here we characterize TRAPPC2L, a novel, highly conserved TRAPP-interacting protein related to TRAPPC2 and the uncharacterized yeast open reading frame YEL048c. TRAPPC2L and TRAPPC2 genes are found in pairs across species and show broad and overlapping expression, suggesting they are functionally distinct, a notion supported by yeast complementation studies and biochemical characterization. RNA interference-mediated knockdown of either TRAPPC2L or TRAPPC2 in HeLa cells leads to fragmentation of the Golgi, implicating both proteins in Golgi dynamics. Gradient fractionation of cellular membranes indicates that TRAPPC2L is found with a portion of cellular TRAPP on very low-density membranes whereas the remainder of TRAPP, but not TRAPPC2L, is found associated with Golgi markers. YEL048c displays genetic interactions with TRAPP II-encoding genes and the gene product co-fractionates with and interacts with yeast TRAPP II. Taken together these results indicate that TRAPPC2L and its yeast ortholog YEL048c are novel TRAPP-interacting proteins that may modulate the function of the TRAPP II complex.

Abstract: X-linked spondyloepiphyseal dysplasia tarda (SEDT-XL) is a skeletal disorder characterized by defective structures of vertebral bodies and/or of epiphyses of the long bones, resulting in moderately short stature and early joint degeneration. TRAPPC2 gene, which is important for collagen secretion, has been reported as causative for SEDT-XL. Here, we report two variants of TRAPPC2 gene of SEDT-XL patients, a missense variant of start codon, c.1A > T, and a deletion variant, c.40delG. To understand molecular consequence of the variants, we establish an in vitro gene expression assay system and demonstrate that both mutated genes are transcribed, but are not properly translated, indicative of the pathogenic nature of those TRAPPC2 variants. In the current study, we provide additional experimental data showing that loss-of-function TRAPPC2 variants are probably causative for SEDT-XL phenotype. These findings further contribute to the understanding the clinical picture related to TRAPPC2 gene.

Abstract: X-linked spondyloepiphyseal dysplasia tarda (SEDT; OMIM 313400) is a rare osteochondrodysplasia that occurs in affected individuals between 3 and 12 yr of age. Clinical features include short trunk, barrel-shaped chest and disproportionate short stature. Radiological abnormalities may become evident between 10 and 14 yr of age and include platyspondyly with hump-shaped central and posterior portions of the vertebrae, narrow disc spaces and moderate epiphyseal dysplasia of the long bones, which may be associated with osteoarthritis. Female heterozygous carriers are clinically and radiographically normal. SEDT is caused by mutations in the TRAPPC2 gene, (trafficking protein particle complex 2), which spans a genomic region of ∼20 kb in Xp22. In a large Chinese SEDT family, we screened all the six exons of the TRAPPC2 gene and identified a novel RNA-splicing mutation (IVS4+1A>G). We also demonstrated that the mutation induced splice pattern change from AT/AC to GT/AG. As a result, the first seven nucleotides of exon 5 were spliced out from the transcript. The prediction of the amino acid sequence showed that the seven nucleotides deletion of the transcript caused frame shift and led to premature translation termination, causing loss of two alpha helices. The results of our study expand the spectrum of the gene mutations associated with SEDT, and will help further to elucidate the role of this protein in the etiology of this form of osteochondrodysplasia. X-linked spondyloepiphyseal dysplasia tarda mutations in the TRAPPC2 gene previously identified as SEDL gene,

Abstract: 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.