Investigation of proteins associated with a VTI13 endosomal trafficking pathway to the lytic vacuole
Conference Year
2023
Abstract
SNAREs (Soluble NSF Attached Receptors) are essential for endosomal trafficking pathways by mediating vesicle fusion between membranes. VTI13 is a member of the VTI family of SNARES in Arabidopsis that localizes to early endosomes and the tonoplast membrane. Genetic analysis showed that vti13 mutants are defective in polarized root hair growth and root epidermal cell wall organization. To identify proteins that are trafficked to the vacuole using a VTI13-dependent pathway, we used a proteomic approach with transgenic lines expressing GFP-VTI13. These experiments identified several proteins involved in Clathrin-mediated endocytosis including Dynamin-Related Proteins (DRP) 1, DRP2A, DRP2B, and Clathrin Heavy Chain (CHC)1 and CHC2. Genetic analysis of drp1, drp2a and drp2b showed that they were defective in root hair growth, supporting a role for these proteins in a VTI13-mediated endosomal trafficking pathway. We also identified several plasma membrane proteins that are potential cargo of VTI13 early endosomes including Patellin 1 (PATL1), Arabidopsis Proton ATPase (AHA)1 and AHA2. AHA1 and AHA2 are the major proton ATPases functioning in root epidermal cells where they act in maintaining a pH gradient necessary for cell expansion. Patellin 1 localizes to the cell plate, associates with phosphatidylinositols in the plasma membrane, and likely functions in diverse aspects of plant growth. I have generated transgenic plants expressing RFP fusions for PATL1, AHA1, and AHA2 that I will cross with GFP-VTI13 expressing lines. We will use F1 seedlings from these crosses to 1) investigate whether PATL1, AHA1, and AHA2 colocalize with GFP-VTI13 in early endosomes and 2) whether they are trafficked to the vacuole in Arabidopsis seedlings. In addition, we are using a genetic approach to identify whether the endosomal trafficking pathway used by VTI13 to traffic cargo to the lytic vacuole is RAB-dependent or RAB-independent in root epidermal cells.
Primary Faculty Mentor Name
Mary Tierney
Status
Graduate
Student College
College of Agriculture and Life Sciences
Second Student College
Graduate College
Program/Major
Plant Biology
Primary Research Category
Life Sciences
Investigation of proteins associated with a VTI13 endosomal trafficking pathway to the lytic vacuole
SNAREs (Soluble NSF Attached Receptors) are essential for endosomal trafficking pathways by mediating vesicle fusion between membranes. VTI13 is a member of the VTI family of SNARES in Arabidopsis that localizes to early endosomes and the tonoplast membrane. Genetic analysis showed that vti13 mutants are defective in polarized root hair growth and root epidermal cell wall organization. To identify proteins that are trafficked to the vacuole using a VTI13-dependent pathway, we used a proteomic approach with transgenic lines expressing GFP-VTI13. These experiments identified several proteins involved in Clathrin-mediated endocytosis including Dynamin-Related Proteins (DRP) 1, DRP2A, DRP2B, and Clathrin Heavy Chain (CHC)1 and CHC2. Genetic analysis of drp1, drp2a and drp2b showed that they were defective in root hair growth, supporting a role for these proteins in a VTI13-mediated endosomal trafficking pathway. We also identified several plasma membrane proteins that are potential cargo of VTI13 early endosomes including Patellin 1 (PATL1), Arabidopsis Proton ATPase (AHA)1 and AHA2. AHA1 and AHA2 are the major proton ATPases functioning in root epidermal cells where they act in maintaining a pH gradient necessary for cell expansion. Patellin 1 localizes to the cell plate, associates with phosphatidylinositols in the plasma membrane, and likely functions in diverse aspects of plant growth. I have generated transgenic plants expressing RFP fusions for PATL1, AHA1, and AHA2 that I will cross with GFP-VTI13 expressing lines. We will use F1 seedlings from these crosses to 1) investigate whether PATL1, AHA1, and AHA2 colocalize with GFP-VTI13 in early endosomes and 2) whether they are trafficked to the vacuole in Arabidopsis seedlings. In addition, we are using a genetic approach to identify whether the endosomal trafficking pathway used by VTI13 to traffic cargo to the lytic vacuole is RAB-dependent or RAB-independent in root epidermal cells.