Reversible cytoplasmic localization of the proteasome in quiescent yeast cells

Abstract

The 26S proteasome is responsible for the controlled proteolysis of a vast number of proteins, including crucial cell cycle regulators. Accordingly, in Saccharomyces cerevisiae, 26S proteasome function is mandatory for cell cycle progression. In budding yeast, the 26S proteasome is assembled in the nucleus, where it is localized throughout the cell cycle. We report that upon cell entry into quiescence, proteasome subunits massively relocalize from the nucleus into motile cytoplasmic structures. We further demonstrate that these structures are proteasome cytoplasmic reservoirs that are rapidly mobilized upon exit from quiescence. Therefore, we have named these previously unknown structures proteasome storage granules (PSGs). Finally, we observe conserved formation and mobilization of these PSGs in the evolutionary distant yeast Schizosaccharomyces pombe. This conservation implies a broad significance for these proteasome reserves.

Intracellular Dynamics of the Ubiquitin-Proteasome-System

“…So far, ~30 subunits of the yeast proteasome were labelled with GFP. All of them reveal the same subcellular localization as thoroughly investigated by direct fluorescence microscopy in living yeast ( Laporte et al , 2008). …”

“…In our species of interest, Saccharomyces cerevisiae , which is an excellent model organism for eukaryotic cells, GFP labelling of proteasomes is achieved by homologous recombination techniques into the chromosomal locus to convert an endogenous proteasomal subunit to a GFP-tagged version ( Enenkel et al , 1999; Laporte et al , 2008; McDonald & Byers, 1997). Nearly all proteasomal genes are essential and could be modified by GFP fusions without interfering with their function; we prefer the CP subunits α4 and β5, the CP-dedicated chaperone Ump1, and the RP subunits Rpn1, Rpt1 and Rpn11 as GFP-labelled reporters, because their GFP fusion proteins are fully incorporated into the proteasomal subcomplexes.…”

“…Later investigations reported a shift towards cytoplasmic proteasomes dependent on the type of the cell line and the density of the cells ( Wojcik & DeMartino, 2003). Proteasome localization also varies with the growth phase in yeast ( Laporte et al , 2008; Weberruss et al , 2013). In growing yeast at logarithmic phase (OD~1), proteasomes are primarily nuclear.…”

“…Through these non-invasive techniques, the localization of the proteasome in growing yeast and highly proliferating cancer cells has been elucidated to be primarily nuclear ( Enenkel et al , 1998; Laporte et al , 2008; McDonald & Byers, 1997; Russell et al , 1999). In line with this finding, increasing evidence in the literature suggests that certain misfolded proteins are imported from the cytoplasm into the nucleus solely for proteasomal degradation ( Park et al , 2013; Prasad et al , 2010).…”

“…PSGs are membraneless organelles which seem to pinch off the NE/ER and are retained as stable entities in the cytoplasm. When cells resume growth, PSGs dissipate and proteasomes are rapidly imported into the nucleus to contribute their function in cell proliferation ( Laporte et al , 2008). The mechanism of PSG formation and clearance is still unknown but seems to be conserved, since PSG-like structures are observed in primary cell lines of non-dividing neuronal cells and in immortalized cell lines of cancer cells, if they are chemically arrested in cell cycle progression ( Kaganovich et al , 2008).…”