Yeast 2-hybrid Testing of β(heavy)-spectrin (geflong) with Candidate Rho and Rab Gtpases and the Re-evaluation of Previous Yeast 2-hybrid

Open Access
Author:
Yeoh, Beng San
Area of Honors:
Biotechnology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Graham Hugh Thomas, Thesis Supervisor
  • Ming Tien, Honors Advisor
  • Wendy Hanna Rose, Faculty Reader
Keywords:
  • Yeast 2-Hybrid
  • Spectrin
  • Rho
  • Rab
  • Fax
  • Eps15
  • Nemy
Abstract:
The spectrin-based membrane skeleton (SBMS) is a network of membrane-associated proteins that also functions as a scaffold for association with other proteins at the plasma membrane. The roles of the SBMS in multicellular animals were initially thought to be restricted to determining cell shape and maintaining membrane integrity. However, current analyses on the SBMS have suggested that the SBMS has a more dynamic role in the cell such as modulating the process of endocytosis, endosomal trafficking and participating in the determination of apical basal polarity. Recently, a study has shown that Rac1 (a Rho GTPase) could be modulated by βH-spectrin, which is in turn regulated by Pak1, an effector for Rac1. In a separate experiment, βH-spectrin was demonstrated to have an epistatic interaction with Rab5. It has also been shown that βH-spectrin plays a role in regulating the progression of the early endosome and decision to either transport protein back to the membrane (recycling), or to lysosomes (degradation). Novel sequence analysis software (GDDA-BLAST) suggests that βH-spectrin contains a Dbl homology domain in its C-terminus. Hence, it is possible that the Dbl homology domain in βH-spectrin may have GEF-like properties including the ability to bind GTPases. So, one hypothesis suggests that βH33 may function as a guanine nucleotide exchange factor (GEF) mimic that binds GTPase proteins and prevents them from being activated by other GEFs. To test this hypothesis, several candidate members from the Rho GTPase family and Rab GTPase family were selected to be tested for direct interaction with GEFlong (a fragment of βH-spectrin C-terminus that contains the predicted Dbl homology domain through the PH domain) by using the yeast 2-hybrid system. In addition, some genes identified in a previous yeast 2-hybrid screen were critically reevaluated.