Cellular growth defects triggered by an overload of protein localization processes

Protein turnover requires cellular resources. However, because resources are finite, ultimate high-level expression of a gratuitous protein potentially leads to overloading and exhaustion of resources1. Ultimate high-level expression of a gratuitous protein, in fact, monopolizes cellular resources for protein synthesis and causes cellular growth defects2,3,4,5,6. In addition to synthesis, protein turnover requires cellular resources for folding, degradation, post-translational modification, and localization. High-level expression of a protein imposes a high demand on these resources and potentially overloads them; for example, high-level expression of an aggregative polyQ-containing protein causes cellular growth defects by sequestering and limiting the chaperone Sis17; disomic yeast strains show growth defects because overexpression of proteins from the extra chromosome overloads the degradation machinery, proteasome8. High-level expression of yellow fluorescent proteins (YFPs) with misfolding mutations cause cellular growth defects9, while a green fluorescent protein (GFP) with a degradation signal has a stronger negative effect on cellular growth than normal GFP10. These proteins may also overload folding and degradation resources when they are highly expressed. For localization of proteins to intracellular compartments, specific types of transport machinery are used. Localization of proteins is usually performed based on the information of localization signals11, and the presence of these signals may be predicted based, in part, on their consensus amino acid sequences. Mitochondrial targeting signals (MTSs) and signal sequences (SSs) located at the N termini of proteins are used to target proteins into the mitochondria and the endoplasmic reticulum (ER), respectively12,13. Nuclear localization signals (NLSs) are used to import proteins into the nucleus14, and nuclear export signals (NESs) are used to export proteins from the nucleus15. The C termini of some proteins contain cytoplasmic membrane-anchoring signals16, and these localization/targeting signals are recognized by specific transport machinery11,17,18,19. Because transport machinery is also a limited cellular resource, high-level expression of a transported protein potentially leads to overload of the transporting process, prevents the transport of other essential proteins, and causes cellular growth defects. However, the overload of localization resources and the physiological consequences of this have never been studied experimentally. The genetic tug of war (gTOW) is a method for estimating the overexpression limit of a protein in yeasts20,21,22. In a gTOW experiment, the limit leading to cellular growth defects is measured as the copy-number limit of the gene encoding the target protein (for details of the gTOW experiment, see Supplementary Method). Previously, we measured the expression limits of a model gratuitous protein, GFP, using the gTOW in the budding yeast Saccharomyces cerevisiae10. We also observed that addition of a degradation signal to GFP reduced the expression limit, probably due to the overloading of degradation resources10. In this study, we attached various localization signals to GFPs and measured their expression limits using the gTOW method. On the basis of these measurements, we evaluated the overloading of localization resources that correspond to various cellular compartments and analyzed the cellular defects triggered by these loads.