Abstract

Rare diseases affect 3.5–5.9% of the global population and mutations in membrane trafficking
proteins are known to cause rare disorders with severe symptoms. The highly conserved transport
protein particle (TRAPP) complexes are key membrane trafficking regulators that are also
involved in autophagy. Pathogenic variants in specific TRAPP subunits are linked to neurological
disorders, muscular dystrophies, and skeletal dysplasias. Characterizing these mutations and
their phenotypes is important for understanding general and specialized roles of TRAPP subunits.
Patient-derived cells are not always available, which poses a limitation for the study of these
diseases. Therefore, other systems, like the yeast Saccharomyces cerevisiae, can be used to
dissect the mechanisms at the intracellular level underlying these disorders. The development of
CRISPR/Cas9 technology in yeast has enabled a scar-less editing method that creates an
efficient humanized yeast model. This project focuses on humanizing the core TRAPP complex
subunits in yeast to generate a platform for studying TRAPP variants associated with disease in
humans. Core yeast subunits were humanized by replacing with their human orthologs and
TRAPPC1, TRAPPC2, TRAPPC2L, TRAPPC6A, and TRAPPC6B were found to successfully
replace their yeast counterparts. This system was used for studying the first reported TRAPPC1
variant identified in humans, which is a compound heterozygous mutation. I show that the
maternal variant (TRAPPC1 p.Val121Alafs*3) is non-functional while the paternal variant
(TRAPPC1 p.His22_Lys24del) is conditional-lethal in yeast. Results show that the paternal
TRAPPC1 variant affects non-selective autophagy in yeast whereas membrane trafficking and
autophagy are defective in patient fibroblasts. This study suggests that humanized yeast can be
an efficient means to study TRAPP subunit variants in the absence of human cells, and it lays the
foundation for characterizing further TRAPP variants through this system.