Yeast ribosomal stalk heterogeneity in vivo shown by two-photon FCS and molecular brightness analysis

The stalk of Saccharomyces cerevisiae ribosomes contains, on average, five distinct proteins, namely P0 and four acidic proteins, P1alpha, P1beta, P2alpha, and P2beta. Each ribosome contains only one copy of P0, but the distribution of the acidic proteins among the ribosome population in vivo has not been determined. Using two-photon fluorescence correlation spectroscopy and scanning FCS, on cells expressing EGFP-tagged P0, P1, and P2 proteins, we show, with brightness analysis, that individual yeast ribosomes in vivo are compositionally heterogeneous in regard to P1alpha, P1beta, P2alpha, and P2beta. These results are relevant to the hypothesis, based on in vitro studies, that the overall cellular pattern of expressed proteins can be determined by the distribution of the stalk proteins among the ribosome population.