resulting in a wide displacement of the PsR
domain. As the pool of free KaiA dimers is depleted, KaiC switches back to autodephosphorylation activity. Complete dephosphorylation of
KaiC results in dissociation of the KaiCBA complex by loss of KaiA2B1 subcomplexes, thereby
completing one cycle of the oscillator. In cyanobacterial cells, KaiC and KaiB are produced from
the same operon and in 10- to 100-fold excess to
KaiA (28). The high excess of KaiCB over free
KaiA could promote efficient sequestration of
KaiA in vivo. The model presented here can thus
serve as a framework to better understand the
circadian clock in cyanobacterial cells.
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We thank J. Andres and M. Yazdanyar for help with protein
expression, O. Mihalache for help with sample preparation, F. Beck
and A. Aufderheide for assistance with image processing, and
C. Benda for help with PHENIX software. This work was supported
by the Netherlands Organisation for Scientific Research (NWO)
Roadmap Initiative Proteins@Work grant 184.032.201 to A.J.R.H.,
the European Union Seventh Framework Programme ManiFold
grant 317371 to A.J.R.H. and P.L., and the German Research
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Materials and Methods
Figs. S1 to S15
Tables S1 to S4
Data S1 to S3
10 June 2016; accepted 13 February 2017
Aggregation of the Whi3 protein, not
loss of heterochromatin, causes
sterility in old yeast cells
Gavin Schlissel,1 Marek K. Krzyzanowski,2 Fabrice Caudron,2,3
Yves Barral,2† Jasper Rine1†
In yeast, heterochromatin silencing is reported to decline in aging mother cells, causing sterility
in old cells. This process is thought to reflect a decrease in the activity of the NAD+ (oxidized
nicotinamide adenine dinucleotide)–dependent deacetylase Sir2. We tested whether Sir2
becomes nonfunctional gradually or precipitously during aging. Unexpectedly, silencing of the
heterochromatic HML and HMR loci was not lost during aging. Old cells could initiate a mating
response; however, they were less sensitive to mating pheromone than were young cells
because of age-dependent aggregation of Whi3, an RNA-binding protein controlling S-phase
entry. Removing the polyglutamine domain of Whi3 restored the pheromone sensitivity of old
cells. We propose that aging phenotypes previously attributed to loss of heterochromatin
silencing are instead caused by aggregation of the Whi3 cell cycle regulator.
Budding yeast divide asymmetrically, and each yeast mother cell produces a finite number of daughter cells in its lifetime. This process—yeast replicative aging—has been studied for insights into aging more
broadly, because the processes that underlie aging
in yeast might be related to factors that underlie
aging in other asymmetrically dividing cells (1).
In Saccharomyces cerevisiae, haploid mother
cells lose the ability to mate as they age (2). It has
been proposed that old mother cells fail to mate
as a consequence of a decline in Sir2 function,
which would cause loss of heterochromatic gene
silencing of the auxiliary mating-type loci HML
and HMR (3). Loss of silencing at HML and HMR
in old cells has been attributed to the redistribu-
tion of Sir proteins to the nucleolus and to a de-
crease in available Sir2 (4–6). Furthermore, old
cells may be either limited for the Sir2 substrate
nicotinamide adenine dinucleotide (oxidized form;
NAD+) or exposed to high concentrations of
nicotinamide (NAM), an inhibitor of Sir2, resulting in the inactivation of Sir2 in old cells
and thus sterility (7–9).
We characterized transcriptional repression by
Sir2 by testing whether transient loss-of-silencing
events at HML might precede the complete loss
of silencing attributed to the oldest cells. To study
silencing at HML in a yeast mother cell, we mon-
itored pedigrees of haploid cells carrying a Cre-
based silencing reporter (10). The reporter uses a
Cre recombinase gene inserted in place of HMLa2
and a fluorescent reporter inserted at a euchromatic
locus elsewhere in the genome (Fig. 1A). Loss of
silencing at hmla2D::CRE induces a permanent
and heritable switch from expressing red fluores-
cent protein (RFP) to expressing green fluorescent
protein (GFP) (Fig. 1A), and the sensitivity of the
Cre reporter approaches the sensitivity of single-
molecule RNA fluorescent in situ hybridization
(10). We manually separated daughter cells from
their mothers to analyze pedigrees in two common
strain backgrounds, S288c and W303, and observed
no loss-of-silencing events in dozens of pedigrees
of haploids, diploids, and hybrids (Fig. 1B).
To measure the frequency of silencing loss as a
function of a cell’s life span, we extended the
pedigree analysis by using a microfluidic device
that traps mother cells and separates their buds.
We analyzed more than 1500 yeast pedigrees at
single-cell resolution and observed 13 loss-of-
silencing events (Fig. 1C and movie S1). Further-
more, we found that a cell’s age did not affect its
ability to maintain silencing of HML, and the
overwhelming majority of yeast mother cells
stopped dividing without even a transient loss of
silencing (Fig. 1D). As a control, when Sir2 ac-
tivity in old cells was inhibited by addition of
NAM, all surviving cells lost silencing, suggesting
that old cells did not accumulate NAM in amounts
that inactivate Sir2 (Fig. 1E and movie S2). Similar
results were obtained by analyzing a GFP gene
inserted in place of HMLa and using an alter-
native microfluidic design, indicating that the
observation was independent of the reporter and
the microfluidic setup used (fig. S1). Previous
studies have shown that Sir2 protein levels de-
crease in cells that are more than seven genera-
tions old; however, we found no evidence of a
decrease in Sir2 activity at HML (4). It is possible
that a decrease in Sir2 levels in old cells could affect
other Sir2 complexes, including the nucleolar RENT
1184 17 MARCH 2017 • VOL 355 ISSUE 6330 sciencemag.org SCIENCE
1Department of Molecular and Cell Biology, University of
California–Berkeley, Berkeley, CA 94720, USA. 2Institute of
Biochemistry, ETH Zürich, Zürich, Switzerland. 3King's
College London, London, UK.
*These authors contributed equally to this work. †Corresponding
author. Email: email@example.com (Y.B.); jrine@berkeley.