To validate whether Any1 is a Retromer cargo
Project Description
The highly conserved Retromer complex mediates the retrieval of specific cargoes from endosomes to the Golgi. These cargoes are concentrated in distinct regions of the endosomal membrane, where tubules form to transport proteins to the Golgi. In yeast, the Retromer complex can be dissected into two subcomplexes: a trimer of Vps35, Vps29 and Vps26 (referred to as CSC) that mediates cargo selection, and a dimer of Vps5 with Vps17 (referred to as SNX) that promotes tubule or vesicle formation. Additionally, Retromer interacts with the yeast PROPPIN Atg18 to form the CROP complex, which is involved in vacuolar membrane fission. Given that Any1 is an endosomal protein that is also found on the Golgi, we hypothesize that Any1 is a novel Retromer cargo.
Our preliminary data show that in Retromer mutant (vps35∆) cells, Any1 was mostly incorrectly sorted in the vacuole lumen, whereas the CROP mutant (atg18Δ) had no effect on Any1 localization. These data suggest that Any1 is a novel Retromer cargo, which facilitates its cycling between the Golgi and endosomes. To further confirm this, we will investigate the direct interaction between Any1 and the Retromer subcomplex CSC. Specifically, we will perform Co-IP of Any1–TAP from wild-type cells and analyze the binding of Vps35-GFP, Vps29-GFP, and Vps26-GFP. In parallel, we will purify 3xFLAG-tagged Any1 from yeast and analyze its interaction with purified CSC using an in vitro pulldown assay.
Our preliminary data show that in Retromer mutant (vps35∆) cells, Any1 was mostly incorrectly sorted in the vacuole lumen, whereas the CROP mutant (atg18Δ) had no effect on Any1 localization. These data suggest that Any1 is a novel Retromer cargo, which facilitates its cycling between the Golgi and endosomes. To further confirm this, we will investigate the direct interaction between Any1 and the Retromer subcomplex CSC. Specifically, we will perform Co-IP of Any1–TAP from wild-type cells and analyze the binding of Vps35-GFP, Vps29-GFP, and Vps26-GFP. In parallel, we will purify 3xFLAG-tagged Any1 from yeast and analyze its interaction with purified CSC using an in vitro pulldown assay.
Supervisor
GAO, Jieqiong
Quota
2
Course type
UROP1000
Applicant's Roles
Our lab is seeking a motivated undergraduate student to join our research team as an Introductory Research Assistant. This position is designed for students with no prior research experience who are eager to learn the fundamentals of scientific inquiry. The successful applicant will gain hands-on experience in the lab.
Applicant's Learning Objectives
This research experience will provide undergraduate students with opportunities to develop their scientific thinking and key skills in scientific inquiry and laboratory techniques. Specifically, students will
(1) Understand the scientific method and its application to the research project.
Students will gain a practical understanding of the scientific method through hands-on experience. This includes learning to construct yeast strains, verify protein expression using Western Blot analysis, purify proteins from yeast, and perform in vitro pulldown assays.
(2)Develop literature searching and review skills.
(3)Understand data management and organization principles.
(1) Understand the scientific method and its application to the research project.
Students will gain a practical understanding of the scientific method through hands-on experience. This includes learning to construct yeast strains, verify protein expression using Western Blot analysis, purify proteins from yeast, and perform in vitro pulldown assays.
(2)Develop literature searching and review skills.
(3)Understand data management and organization principles.
Complexity of the project
Easy