Abstract

The Transport Protein Particle (TRAPP) complex is a highly conserved multi-subunit tethering complex that plays a critical role in membrane trafficking. Mutations in TRAPP complex subunits have been implicated in a growing spectrum of rare genetic disorders, yet the molecular mechanisms underlying variant pathogenicity often remain unclear. In this thesis, I developed a humanized yeast platform to enable systematic functional characterization of TRAPP complex variants of unknown significance. Using a stepwise gene replacement strategy in Saccharomyces cerevisiae, I reconstructed the core TRAPP complex with human orthologs, and constructed a strain containing five of the human subunits. The integration of human subunits was validated through RT-PCR and Western blotting. Growth assays revealed that partial humanization of the core complex recapitulates key functional aspects of TRAPP assembly and enables the investigation of disease-associated variants in vivo. Structural modeling and clash analysis using PyMOL provided insights into the impact of specific mutations on complex stability and subunit interactions. Introduction of a clinically relevant C3 (L131F) variant into the humanized strain resulted in pronounced growth defects and predicted structural clashes, supporting its pathogenicity. This work demonstrates the power of humanized yeast as a model for elucidating genotype-phenotype relationships in rare TRAPPopathies, and provides a versatile platform for variant interpretation, mechanistic studies, and potential therapeutic screening.