Identification of Proteins that Interact with Patellin1 Using a Yeast Two-Hybrid System

Elizabeth Le
Wellesley College

Patellin1 (PATL1), a novel protein isolated in the Peterman lab during a biochemical screen for plant F-actin-binding proteins, is hypothesized to play a role in membrane trafficking during plant cytokinesis. Previous work done on PATL1 has shown that it appears at the cell plate during telophase and persists after cytokinesis. It is also found in association with Golgi-size structures during interphase and metaphase. Sequencing of PATL1 has uncovered a Sec14 homologous domain, a coiled-coil region and a GOLD domain (Peterman et al., 2004). These domains suggest a role for PATL1 in vesicle trafficking during cytokinesis. Because secretion and trafficking require multiple proteins, identification of proteins that interact with PATL1 using a yeast two-hybrid screen may give clues about its role in cytokinesis. A Gal4 transcription factor with two separate domains, a DNA-binding domain fused to a PATL1 N-terminal segment and a transcription activating domain fused to an Arabidopsis root and leaf cDNA library, is being used. Transcriptional activation of the HIS3, ADE2 and MEL1 reporter genes due to the reconstitution of Gal4 in the yeast strain AH109 has identified Arabidopsis cDNAs encoding proteins that may interact with PATL1. Recovery of these interacting Arabidopsis cDNAs will allow for the identification of proteins that interact with PATL1, and will shed more light on its function in cytokinesis.