Using Drosophila S2 Cells to Measure S phase-Coupled Protein Destruction via Flow Cytometry

Abstract

Cell proliferation depends on the timely synthesis and destruction of proteins at specific phases of the cell cycle. Recently it was discovered that the destruction of several key cell cycle regulatory proteins during S phase is coupled directly to DNA replication. These proteins harbor a motif called a PIP degron that mediates binding to chromatin bound PCNA at replication forks and recruits the CRL4Cdt2 E3 ubiquitin ligase. These interactions comprise an elegant mechanism for coupling DNA replication with ubiquitylation and subsequent proteolysis by the 26S proteasome. Here we describe a flow cytometry-based method using Drosophila S2 cells that recapitulates S phase-specific protein proteolysis. Because of the high degree of evolutionary conservation of the PIP degron and CRL4Cdt2 and the ease of culturing and inhibiting gene function by RNAi in S2 cells, our flow cytometric method should serve as a general tool for determining whether any eukaryotic protein is subject to replication-coupled protein destruction.