Identification of the synthetic lethal mutations in yeast expressing a C-terminal truncation of the delta subunit (Ret2p) of COPI coatomer

Abstract

In the secretory pathway in Saccharomyces cerevisiae, the retrograde transport from Golgi to the ER is mediated by COPI coated vesicles. The COPI coat is compromised of the heptameric complex (α-COP, β-COP, γ-COP, δ-COP, ε-COP and ζ-COP), termed coatomer, and the small GTPase Arf1. SNARE proteins are consist of a subset of vesicle cargo proteins and are important for specific vesicle fusion event. However, less is known for the interaction between the coatomer δ-COP (Ret2p) and the SNAREs. Sec22p is one of the SNAREs cycle between the ER and the Golgi apparatus. Previous data has show that amino acid substitutions in Sec26p/β-COP and Ret2p/δ-COP disrupt Sec22p’s Golgi localization. I obtained eight ret2 mutants that are defective in Sec22p retention and that are not temperature sensitive for growth. Surprisingly, a number of these mutants contained nonsense mutations (stop codons) in the ret2 coding region. These mutants indicate that the C-terminus of Ret2p is not essential for cell growth. To further investigate the requirement of the C-terminus of Ret2p, I generated incremental C-terminal truncations of the protein. Among the ret2 truncation mutants, ret2 (amino acids 1-120) cells were temperature-sensitive for growth and also significantly reduced steady-state levels of Ret2p relative to other RET2 mutants – as judged by immunoblotting. Based on these results, I speculate that Ret2p C-terminus (171a.a.-546 a.a.) is not essential for cell growth and the amino acids 120-170 of Ret2p are important for the essential function of Ret2p. In eukaryotic cells, the length and amino acid composition of the C-terminus of δ-COP (RET2) is evolutionarily conserved and the RET2 gene is essential for yeast cell growth. To gain deeper insight into the role of the C-terminus of Ret2p a synthetic lethal screen was carried out using with one of the truncations - ret2p ^(1-170). From the synthetic lethal screen I identified the GLO3 gene. Glo3p encodes a G̲TPase a̲ctivating p̲rotein (GAP) for the COPI GTPase Arf1p. Moreover, interactions between Glo3p and COPI coat (coatomer), vesicle cargos and SNAREs have been reported. In addition to its GAP domain, Glo3p also contains a cargo-binding domain termed BoCCS (Binding of Coatomer, Cargo and SNAREs) as well as the Glo3p-regulatory motif (GRM). Using site-directed mutagenesis and complementation analysis in yeast I established that the synthetic lethal relationship between ret2p^(1-170) and GLO3 results from the loss of Glo3p’s GAP activity.

Date of Award	2013
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology