Investigating the Interaction Between Any1 and Retromer
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 facilitate transport to the Golgi. In yeast, the Retromer complex comprises two subcomplexes: the cargo-selecting complex (CSC; Vps35, Vps29, Vps26) and the tubule/vesicle-forming complex (SNX; Vps5, Vps17). Furthermore, Retromer interacts with the yeast protein Atg18 (PROPPIN) to form the CROP complex, which participates in vacuolar membrane fission. Given that Any1 is an endosomal protein also localized to the Golgi, we hypothesize that Any1 is a novel Retromer cargo.
Preliminary data demonstrate that in retromer mutant (vps35Δ) cells, Any1 mislocalizes to the vacuole lumen, whereas the CROP mutant (atg18Δ) does not affect Any1 localization. These findings suggest that Any1 is a Retromer cargo, facilitating its cycling between the Golgi and endosomes. To confirm this, we will investigate a direct interaction between Any1 and the CSC subcomplex. Specifically, we will perform co-immunoprecipitation (Co-IP) of Any1-TAP from wild-type cells and analyze 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.
Preliminary data demonstrate that in retromer mutant (vps35Δ) cells, Any1 mislocalizes to the vacuole lumen, whereas the CROP mutant (atg18Δ) does not affect Any1 localization. These findings suggest that Any1 is a Retromer cargo, facilitating its cycling between the Golgi and endosomes. To confirm this, we will investigate a direct interaction between Any1 and the CSC subcomplex. Specifically, we will perform co-immunoprecipitation (Co-IP) of Any1-TAP from wild-type cells and analyze 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
UROP1100
Applicant's Roles
Our lab is seeking a motivated undergraduate student to join our research team as an Introductory Research Assistant. This position is ideal for students with limited research experience who are eager to learn fundamental scientific techniques and contribute to ongoing research projects. The successful applicant will gain valuable hands-on laboratory experience.
Applicant's Learning Objectives
This research experience will provide undergraduate students with opportunities to develop critical scientific thinking and essential laboratory skills. Participants will:
(1) Master Core Laboratory Techniques: Students will gain hands-on experience applying the scientific method, including yeast strain construction, verification of protein expression via Western blot analysis, protein purification from yeast, and in vitro pulldown assays.
(2) Cultivate Research & Analytical Skills: Students will develop proficiency in literature searching, critical review of scientific publications, and principles of data management and organization.
(3) Understand the Scientific Process: Students will apply the scientific method directly to our ongoing research project, contributing to experimental design, data analysis, and interpretation.
(1) Master Core Laboratory Techniques: Students will gain hands-on experience applying the scientific method, including yeast strain construction, verification of protein expression via Western blot analysis, protein purification from yeast, and in vitro pulldown assays.
(2) Cultivate Research & Analytical Skills: Students will develop proficiency in literature searching, critical review of scientific publications, and principles of data management and organization.
(3) Understand the Scientific Process: Students will apply the scientific method directly to our ongoing research project, contributing to experimental design, data analysis, and interpretation.
Complexity of the project
Easy