Using fluorescent reporters in conjunction with cytometry and statistics to assess nuclear accumulation of ribosomal proteins

Abstract

Fluorescently labeled ribosomal proteins can be used to detect and monitor the intracellular localization of these proteins. Both Rps2, a subunit of the 40S ribosome, and Rpl25, a subunit of the 60S ribosome, have been fused to the coding sequence of GFP at their C-termini and the fusions have been used to monitor their localization within cells using fluorescent microscopy. Normally these proteins are efficiently incorporated into ribosomes and exported into the cytoplasm where they exhibit a low uniform fluorescence. However, these Rps2- and Rpl25-GFP proteins accumulate in the nucleus of mutants defective in ribosome biogenesis or export. Here, we describe a single-cell quantitative method to assess the nuclear accumulation of ribosomal proteins using cytometry and biostatistics. This assay was developed for use with GFP reporters for ribosome biogenesis in budding yeast but could be adapted for use with any GFP reporter that accumulates into the nucleus under adverse conditions.