Subcellular Compartmentalization of Human Ribosomal Protein S6 Turnover

Abstract

The ribosome assembles in the nucleolus of the cellular nucleus. The mechanism of Ribosomal Protein S6 (RPS6) is well understood in the context of the ribosome; however, its function outside the translational machinery remains unknown. Here, we use a nuclear export sequence (NES) to determine the presence and function of RPS6 in the cytoplasm, outside the ribosome. Using restriction ligation cloning and subsequent site-directed mutagenesis, plasmid vectors were constructed that encode a fusion protein of RPS6, pmTurquoise (a Green Fluorescent Protein derivative), and NES. Control plasmids were constructed with and without RPS6 and NES. Western blot analysis confirmed expression of plasmid constructs in human embryonic kidney cells (HEK293), with RPS6-NES-pmTurq detected at 48 hours post-transfection. Nuclear-cytoplasmic preparations were obtained from the tranfected HEK293 cells, which confirmed the presence of both RPS6-mTurq and RPS6-mTurq-NES, in both the nuclear and cytoplasmic fractions, suggesting that the amount of transfected plasmid, and therefore protein overexpressed, overwhelmed the transport machinery. However, RPS6-pmTurq was detectable by Western blot with and without an NES, indicating a stable protein in both cellular compartments. Future experiments will aim to optimize transfection conditions for construct expression and immunopurification, and, together with fluorescent microscopy, confirm the presence and localization of the fusion proteins, their protein interactions, and their post-translational modifications. These studies aim to determine the cytosolic events that contribute to RPS6's extra-ribosomal functions, stability, and transport before its incorporation into the ribosome.