Curing of yeast [PSI+] prion by guanidine inactivation of Hsp104 does not require cell division.

Abstract

Propagation of the yeast prion [PSI+], a self-replicating aggregated form of Sup35p, requires Hsp104. One model to explain this phenomenon proposes that, in the absence of Hsp104, Sup35p aggregates enlarge but fail to replicate thus becoming diluted out as the yeast divide. To test this model, we used live imaging of Sup35p-GFP to follow the changes that occur in [PSI+] cells after the addition of guanidine to inactivate Hsp104. After guanidine addition there was initially an increase in aggregation of Sup35p-GFP; but then, before the yeast divided, the aggregates began to dissolve, and after approximately 6 h the Sup35-GFP looked identical to the Sup35-GFP in [psi+] cells. Although plating studies showed that the yeast were still [PSI+], this reduction in aggregation suggested that curing of [PSI+] by inactivation of Hsp104 might be independent of cell division. This was tested by measuring the rate of curing of [PSI+] cells in both dividing and nondividing cells. Cell division was inhibited by adding either alpha factor or farnesol. Remarkably, with both of these methods, we found that the rate of curing was not significantly affected by cell division. Thus, cell division is not a determining factor for curing [PSI+] by inactivating Hsp104 with guanidine. Rather, curing apparently occurs because Sup35-GFP polymers slowly depolymerize in the absence of Hsp104 activity. Hsp104 then counteracts this curing possibly by catalyzing formation of new polymers.

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Curing of yeast [PSI?] prion by guanidine inactivation
of Hsp104 does not require cell division
Yue-Xuan Wu*, Lois E. Greene*, Daniel C. Masison†, and Evan Eisenberg*‡
*Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8017; and†Laboratory of
Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0851
Communicated by Reed B. Wickner, National Institutes of Health, Bethesda, MD, July 27, 2005 (received for review May 25, 2005)
Propagation of the yeast prion [PSI?], a self-replicating aggregated
form of Sup35p, requires Hsp104. One model to explain this
phenomenon proposes that, in the absence of Hsp104, Sup35p
aggregates enlarge but fail to replicate thus becoming diluted out
as the yeast divide. To test this model, we used live imaging of
Sup35p-GFPtofollowthechangesthatoccurin[PSI?]cellsafterthe
addition of guanidine to inactivate Hsp104. After guanidine addi-
tion there was initially an increase in aggregation of Sup35p-GFP;
but then, before the yeast divided, the aggregates began to
dissolve, and after ?6 h the Sup35-GFP looked identical to the
Sup35-GFP in [psi?] cells. Although plating studies showed that the
yeastwerestill[PSI?],thisreductioninaggregationsuggestedthat
curing of [PSI?] by inactivation of Hsp104 might be independent of
cell division. This was tested by measuring the rate of curing of
[PSI?] cells in both dividing and nondividing cells. Cell division was
inhibited by adding either alpha factor or farnesol. Remarkably,
with both of these methods, we found that the rate of curing was
not significantly affected by cell division. Thus, cell division is not
a determining factor for curing [PSI?] by inactivating Hsp104 with
guanidine. Rather, curing apparently occurs because Sup35-GFP
polymers slowly depolymerize in the absence of Hsp104 activity.
Hsp104 then counteracts this curing possibly by catalyzing forma-
tion of new polymers.
P
a fibrous polymer-like aggregate rich in ?-sheet secondary
structure (1). Normally folded mammalian prion protein has a
high ?-helical content, but its aggregated form contains much
more ?-sheet and is highly resistant to proteolysis. When this
autocatalytic conversion occurs on a large scale in the central
nervous system, the amyloid form accumulates and is associated
with the neurodegeneration in fatal diseases such as scrapie in
sheep, bovine spongiform encephalopathy in cows, and
Creutzfeld–Jacob disease in humans (2). Other proteins form
amyloid in an autocatalytic manner, but only prion protein is
infectious.
Prions also occur in yeast (3). The best-characterized yeast
prion proteins are Sup35p, a translation termination factor, and
Ure2p, which is involved in nitrogen metabolism (4). More
recently,Rnq1pwasidentifiedasaprionprotein,andNew1pwas
shown to contain a domain that can substitute for the prion-
determining region of Sup35p (5–7). Amyloidogenic yeast prion
proteins have an Asn?Gln-rich prion-forming domain that, like
mammalian prion protein, can be converted to a ?-sheet-rich,
protease-resistant conformation in an autocatalytic manner.
When yeast cells containing soluble Sup35p, for example, are
mated to cells containing the prion form of Sup35p, the soluble
form is converted to the aggregated form, which then continues
to propagate as the yeast divide. The term [PSI?] refers to the
Sup35p prion as well as to yeast cells propagating the prion form
of Sup35p. Normally, Sup35p is soluble, and the yeast are
referred to as [psi?].
Although almost nothing is known about the requirement of
accessory proteins for prion propagation in mammals, genetic
experiments have shown that propagation of amyloidogenic
rions (infectious proteins) are proteins that are normally
soluble but autocatalytically propagate as amyloid, which is
yeast prions requires Hsp104, a member of the ClpB class of
protein chaperones that form ring-shaped polymers and disas-
semble protein aggregates (5, 8–10). Paradoxically, high levels of
Hsp104 also convert [PSI?] to [psi?], but this effect is unique for
[PSI?] and does not occur with the other yeast prions. In vitro,
Hsp104 has a dual function in that it both facilitates formation
of Sup35p aggregates and breaks up Sup35p aggregates (11, 12).
The mechanism by which Hsp104 maintains [PSI?] is not yet
understood. Lindquist and colleagues (11, 13) have suggested
that the ability of Hsp104 both to nucleate Sup35p aggregation
and to break up these aggregates plays a role in maintaining
[PSI?]. On the other hand, others have suggested that Hsp104 is
required only to break up aggregates to replicate them so that
they are passed on to daughter cells when yeast divide (14–16).
It was further proposed that Hsp104 breaks up only the small
aggregates, whereas large aggregates form dead-end complexes
unable to propagate the prion trait (15, 17–19).
Investigations into the mechanism of action of Hsp104 have
been facilitated by experiments showing that Hsp104 in vivo can
be inactivated by treating yeast with low levels of guanidine-
hydrochloride (Gdn) (20–22). Whereas it was demonstrated
many years ago that such treatment cures [PSI?], recently it was
shown that Gdn acts by inhibiting Hsp104 ATPase activity (23,
24). Because Gdn inactivates Hsp104 immediately, kinetics of
[PSI?] curing after this inactivation can be monitored straight-
forwardly. When growing cells are exposed to Gdn there is a lag
phaseduringwhichnocuringoccursfollowedbyalinearincrease
in the number of cured cells over time (25).
These kinetic studies have supported the model that Hsp104
acts by breaking up prion polymers, thereby generating new
prion ‘‘seeds.’’ During the lag phase that occurs after Hsp104
activity is inhibited, the prion polymers or seeds are thought to
grow in size but, failing to replicate, become diluted among the
increasing number of dividing cells. Once the number of seeds
percellissufficientlylow,furthercelldivisionproducesdaughter
cells lacking seeds, which are therefore [psi?]. Curing also
appears to require cell division because it does not occur during
stationary phase. In fact, if Gdn-treated cells enter stationary
phase before becoming [psi?], curing of [PSI?] is interrupted.
In our previous studies of yeast expressing a full-length
Sup35-GFP fusion protein (NGMC), we found that NGMC
appeared diffuse in both [psi?] and [PSI?] cells (26). When we
measuredtherateoffluorescencerecoveryafterphotobleaching
(FRAP), however, the rate of recovery of the NGMC fluores-
cence in [PSI?] cells was significantly slower than in [psi?] cells.
This reduced rate reflects the presence of small NGMC aggre-
gates in the [PSI?] cells. This ability to detect NGMC aggregates
allows observation of the seeds thought to be responsible for
Freely available online through the PNAS open access option.
Abbreviations: FRAP, fluorescence recovery after photobleaching; Gdn, guanidine-hydro-
chloride; YPD, yeast extract?peptone?dextrose.
‡To whom correspondence should be addressed at: Laboratory of Cell Biology, National
Heart, Lung, and Blood Institute, 50 South Drive, Room 2525, MSC 8017, Bethesda, MD
20892-8017. E-mail: eisenbee@nhlbi.nih.gov.
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[PSI?] propagation and, thereby, the change that occurs when
Hsp104 activity is inhibited by Gdn.
In this study, we monitored Sup35p aggregation by using
real-time imaging of yeast expressing NGMC and followed the
changes after addition of Gdn. Our results showed that curing
cells of [PSI?] by Hsp104 inactivation does not require cell
division, which has important implications regarding current
understanding of the dynamics of Sup35p aggregates in [PSI?]
cells.
Materials and Methods
Strains, Plasmids, Media, and Growth Conditions. Yeast strains were
[PSI?] and [psi?] derivatives of strain 780-1D (MATa kar1-1
SUQ5 ade2-1his3?202
sup35::KanMX?pJ510 or pJ533) described in ref. 26. Plasmids
pJ510 and pJ533, described in ref. 26, are single-copy plasmids
with the LEU2 marker and NGMC or unmodified SUP35,
respectively. Selection for plasmid maintenance is not necessary
because Sup35p is essential. YPD medium contains 0.5% yeast
extract,2%peptone,and2%dextrose.Culturesweremaintained
in log phase by periodic dilution with fresh medium. For
monitoring NGMC in growing cells, yeast were grown at 30°C on
synthetic medium (SD; 0.7% yeast nitrogen base, 2% glucose)
with complete supplement mixture (CSM; 25 mg?liter adenine,
histidine, leucine, tryptophan, and uracil). This medium has no
autofluorescence and is suitable for imaging living cells by
fluorescence microscopy. To arrest cell division, cells were
treated with 50 ?M ?-factor (Zymo Research) or 100 ?M
farnesol (Sigma). This latter treatment arrests cell division by
inhibiting DNA ligase and histone acetyltransferase (27).
leu2?1trp1?63ura3-52
[PSI?] Curing. Gdn was added to growing cultures to a concen-
tration of 5 mM. The presence of [PSI?], detected by its ability
to suppress the ade2-1 allele in our strains (28), was monitored
by plating culture aliquots on 1?2YPD. On these plates, [PSI?]
(suppressed) cells are white and [psi?] (nonsuppressed) cells are
red. Growth was monitored by optical density (OD600). When
assaying [PSI?] curing in the presence of ?-factor (Zymo Re-
search), the cells were plated and counted because OD600was
not an accurate measure of cell division.
Confocal Microscopy. To immobilize cells for imaging, two-well
chamber 25-mm2coverslips (Lab-Tek) were pretreated with 2
mg?ml Con A (Sigma-Aldrich) for 10 min. Cells were imaged on
a Zeiss LSM 510 confocal microscope and photobleached as
described in ref. 26. For each condition, 15–20 cells were
photobleached. After normalizing each bleach experiment by
setting the maximum fluorescence to 100% and the minimum to
0%, the data were compiled and used to calculate averages and
standard deviations for each condition.
Results
Live cell imaging was used to monitor the changes that occurred
in NGMC after addition of Gdn to [PSI?] cells that had been
transferred directly from a YPD plate to a coverslip for imaging.
We first noted that, before Gdn addition, in contrast to our
previous study in which the NGMC appeared completely diffu-
sive, the NGMC had a granular appearance. Then, as shown in
Fig. 1A, in the first hour after Gdn addition, the NGMC granules
clearly coalesced, which, in about one-third of the cells, led to the
formation of a single granule. However, remarkably, with longer
incubation times all of the granules began to dissolve so that at
Fig.1.
incubating with 5 mM Gdn for the indicated times. Fluorescence and differential interference contrast images are of the same fields. DAPI staining showed that
the granules were not in the nucleus. (Scale bar, 2 ?m.) (B) FRAP of NGMC in [PSI?] cells measured prior and after Gdn treatments. (C) Cells first treated with Gdn
for6h(filledtriangles)andthentransferredtomediumwithoutGdnfor2h(opentriangles).FRAPofNGMCin[psi?]cellsisshownforcomparison(filledcircles).
DynamicsofaggregateformationafterGdntreatment.(A)NGMCin[PSI?]cellswasimagedbyusingconfocalfluorescencemicroscopybeforeandafter
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2 h there was significantly less granularity. This dissolution of the
NGMC granules continued with further incubation with Gdn so
that at 3 h the NGMC appeared diffuse in most cells. FRAP
studies on NGMC also showed a biphasic response after Gdn
treatment (Fig. 1B). One hour after Gdn treatment, although the
NGMC in the large granules was completely immobilized (data
notshown),thecytosolsurroundingtheNGMCgranulesshowed
a slower rate of fluorescence recovery compared with the rate
observed before Gdn addition. But after 5 h of incubation in
Gdn, the rate of FRAP had again increased, becoming identical
to the rate observed in [psi?] cells. Note that the extent of
fluorescence recovery was only ?50% in these experiments
because photobleaching caused a decrease in the total fluores-
cence of the NGMC in the cells.
Because the fluorescence of [PSI?] cells used in these studies
was different from that observed in our previous studies in which
thediffuseappearanceofNGMCwassimilarin[PSI?]and[psi?]
cells, we investigated the effect of growth conditions on the
appearance of NGMC in [PSI?] cells. We found that NGMC was
much more granular in [PSI?] cells in late log phase than in early
log phase where the NGMC was completely diffuse. Therefore,
for the remainder of our studies, cells were always kept in early
log phase unless otherwise noted.
In examining the changes that occurred upon addition of Gdn
to [PSI?] cells in early log phase, we found that Gdn treatment
first caused the initially diffuse NGMC to become slightly
aggregated with the appearance of small granules occurring over
the first hour (see Fig. 6A, which is published as supporting
information on the PNAS web site). Then, with further incuba-
tion with Gdn, the NGMC again became diffuse. Similarly, the
FRAP studies showed that there initially was a slight decrease
in the rate of fluorescence recovery after Gdn treatment,
followed by an increase in this rate until it became identical to
that of NGMC in [psi?] cells (see Fig. 6B). As expected, Gdn had
no effect on the FRAP rate of NGMC in [psi?] cells (see Fig.
6C). Therefore, the addition of Gdn to [PSI?] cells in early log
phase caused essentially the same biphasic effect on NGMC
aggregation that occurred with [PSI?] cells in late log phase,
although the results were much more dramatic with the latter
cells than with the former.
Even though NGMC in [PSI?] cells showed a [psi?] FRAP
rate after 6 h of Gdn treatment, it is unlikely that these cells had,
infact,become[psi?]becausebyplating,wefoundthattherewas
no significant curing of [PSI?] during the first 10 h of Gdn
incubation (Fig. 2). Therefore, it is not surprising that, when Gdn
was removed after 6 h of treatment, the rate of fluorescence
recovery once again slowed, returning over a period of ?2 h to
the rate observed in the [PSI?] cells before Gdn treatment (Fig.
1C). Nevertheless, the observation that Gdn significantly re-
duced NGMC aggregation in mother cells as well as daughter
cells raised the possibility that, over a longer period, Gdn could
cure [PSI?] cells without cell division occurring.
To test whether cell division was indeed necessary for curing
of [PSI?], we followed the kinetics of [PSI?] curing in the
presence and absence of ?-factor by periodically removing
aliquots of the Gdn-treated cultures and plating cells. As ex-
pected, without Gdn the yeast cells remained 100% [PSI?] over
a period of 40 h both in the presence and absence of ?-factor
(Fig. 2 A and B). An example of this plating is shown in Fig. 2B
Inset, which shows the curing at 43 h, as indicated by the red
colonies. Therefore, neither the presence of ?-factor nor the
absence of cell division affected [PSI?] stability. Without ?-fac-
tor, the time course of conversion of [PSI?] to [psi?] caused by
Gdn was very similar to what we and others observed previously
(25, 28). After a lag period of ?12 h, there was a steady decrease
in the fraction of [PSI?] cells until almost all of the cells were
[psi?] at ?43 h. Remarkably, however, when this same experi-
ment was carried out in the presence of ?-factor, where almost
no cell division occurred, the time course of conversion from
[PSI?] to [psi?] was very similar. In this case, there was a lag of
?15 h, followed by the complete disappearance of [PSI?] after
?43 h (Fig. 2, open triangles). Experiments were performed to
test the viability of cells treated with ?-factor and Gdn by
determining cell density with a hemocytometer, followed by
plating. The results showed that cells treated with ?-factor and
Gdn for 40 h were ?80% viable. Furthermore, using fluores-
cence, we found that the level of NGMC was not significantly
changed during the 40-h incubation period with ?-factor and
Gdn.
Essentially identical results were obtained when cell division
wasreversiblyarrestedbytreatmentwithfarnesol(27).Asshown
in Fig. 3A, farnesol-treated cells showed the same rate of
conversion from [PSI?] to [psi?] as the control cells. However,
the cell division of the farnesol-treated cells was much slower
than the control cells, especially during the first 25 h, during
which the farnesol-treated cells divided only three times while
the control cells went through 13 generations (Fig. 3B). Finally,
wefoundthatthesamechangesinNGMCfluorescenceoccurred
when Gdn was added to [PSI?] cells in the presence of ?-factor
as in its absence (see Fig. 7, which is published as supporting
information on the PNAS web site). Therefore, it appears that
the conversion of [PSI?] to [psi?] by inhibition of Hsp104 occurs
Fig. 2.
arrested by ?-factor. (A) [PSI?] cells were cultured in SD?CSM with the
indicated additions of Gdn and ?-factor. Curing of [PSI?] was assessed by
plating on 1?2YPD. Each time point represents an average of two indepen-
dentexperiments.(B)GrowthratesofcellsinA.(Inset)Exampleofcolonieson
1?2YPD arising from cells of a 43-h sample. ?-F, ?-factor.
Gdn curing of [PSI?] is independent of cell division when division is
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not only without cell division but also, more remarkably, is not
significantly affected by cell division.
One line of supporting evidence that inhibition of Hsp104, by
itself, cannot convert yeast from [PSI?] to [psi?] without cell
division is that Gdn does not induce this conversion in station-
ary-phase cells (25). We monitored stationary-phase [PSI?] cells
by fluorescence microscopy and plating before and after adding
Gdn. As we observed previously (26), when [PSI?] cells express-
ing NGMC were in stationary phase, the NGMC aggregates into
numerous visible fluorescent foci. During a 72-h period of
exposure to Gdn, the cells retained these large foci, and the
overall fluorescence did not change (Fig. 4). Furthermore,
throughout this time period the cells remained 100% [PSI?] as
determined by periodic plating. Therefore, Gdn had no effect on
NGMC fluorescence in stationary cells, and as was observed for
unmodified Sup35p, Gdn alone could not convert cells express-
ing NGMC from [PSI?] to [psi?] when the cells were in
stationary phase.
Another line of evidence suggesting that cell division is
required for loss of [PSI?] is that the addition of ethanol to
Gdn-treated [PSI?] cells slows both growth rate and rate of
conversion from [PSI?] to [psi?] to the same extent (25). Thus,
it appears that there is a quantitative relationship between how
rapidly cells divide and how rapidly [PSI?] is lost. In contrast, if
our observation that cell division is not required for [PSI?] loss
is correct, it would imply that the effect of ethanol is unrelated
to its effect on growth rate. We therefore tested the effect of
ethanol on both cell division and the time course of conversion
of [PSI?] to [psi?], both in the presence and absence of ?-factor
(Fig. 5). To make a more direct comparison with earlier exper-
iments, we used yeast cells expressing unmodified Sup35p.
In agreement with the results of Eaglestone et al. (25), we
indeed observed that ethanol doubled the yeast cell division time
(Fig. 5B, open squares). Similarly, we found that the presence of
ethanol caused a doubling in the lag time that occurred before
[PSI?] began to linearly decrease after Gdn treatment. However,
Fig. 3.
arrested by farnesol. (A) Curing of [PSI?] cells with the indicated additions of
Gdn and farnesol. Each time point represents average of two independent
experiments. (B) Growth rates of cells in A. (Inset) Example of colonies from
cells in an aliquot of a 43-h sample. FOH, farnesol.
Gdn curing of [PSI?] is independent of cell division when division is
Fig. 4.
to stationary phase cells, NGMC was examined periodically over 72 h by
confocal fluorescence microscopy. Shown are cells before and 44 and 72 h
after adding Gdn.
Gdn cannot cure stationary phase [PSI?] cells. After addition of Gdn
Fig. 5.
reduced rate of cell division caused by ethanol. (A) [PSI?] cells expressing
native Sup35p were grown under the indicated conditions, and [PSI?] curing
was monitored by plating. (B) Growth rates of the cultures shown in A. ?-F,
?-factor; EtOH, ethanol.
Delayed Gdn curing of [PSI?] by ethanol (3%) is unrelated to the
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ethanol also doubled this lag time in the presence of ?-factor
(Fig. 5A, filled squares), even though essentially no cell division
occurred under this condition (Fig. 5B). Therefore, the delay in
the Gdn-induced conversion of [PSI?] to [psi?] caused by
ethanol is apparently unrelated to its effect on cell division.
Discussion
We used our previously described full-length GFP construct of
Sup35p to investigate the mechanism of curing [PSI?] by inhib-
iting Hsp104 activity with Gdn. After adding Gdn to growing
yeast, we saw a biphasic response first with an increase in the
aggregation of NGMC, and then with a dissolution of the
aggregates resulting in their disappearance over a period of
?5–6 h. These changes in the aggregation state of NGMC were
independent of cell division. Therefore, our data contradict the
possibility that Gdn curing of [PSI?] occurs because mother cells
retain large NGMC aggregates that are not passed on to
daughter cells during cell division thus causing daughters to
become [psi?].
The dissolution of NGMC aggregates that began after 1 h in
both mother and daughter cells showed that, even when Hsp104
activity is inhibited, NGMC aggregates undergo dissolution.
Furthermore, this dissolution was not limited to large NGMC
granules. Even after the large aggregates dissolved, our FRAP
analysis showed that the level of NGMC aggregation continued
to decrease over the next 3–4 h until the FRAP became identical
to that observed for [psi?] cells. However, despite having [psi?]
FRAP kinetics, these cells were still [PSI?], reverting to the
slower [PSI?] FRAP after Gdn was removed. It should be noted
that preliminary studies using both FRAP and fluorescence
correlation spectroscopy suggest that NGMC in [psi?] cells is
much larger than GFP perhaps because even monomeric Sup35p
is associated with ribosomes or other proteins in the cytosol.
Therefore, even when the FRAP becomes identical to that
observed in the [psi?] cells, the Sup35p may still be aggregated,
which could explain why, despite having [psi?] FRAP, cells can
still be [PSI?].
The steady decrease in aggregation during the 6 h after
inactivating Hsp104 occurred in both mother and daughter cells,
suggesting that cell division is not involved in this phenomenon.
However, as Tuite and his colleagues proposed (15), it still
seemed possible that the decreased level of aggregation was
caused by cell division gradually diluting out NGMC aggregates
too small to be detected by fluorescence methods. Remarkably,
however, nondividing ?-factor- or farnesol-treated cells began to
become [psi?] after a lag of ?10 h, the same time course seen
by us and others for Gdn curing of [PSI?] in dividing cells. These
data imply that the decrease in NGMC aggregation, which we
observed by FRAP during the first 5 h, continued as long as Gdn
was present, whether or not cell division occurred, until all
[PSI?] seeds were eliminated.
The same phenomenon occurred in the presence of ethanol,
which caused the same decrease in the rates of cell division and
of [PSI?] curing (25). However, this correlation seems to be
fortuitous because we showed that ethanol caused the same
decrease in the rate of [PSI?] curing when cell division was
arrested by ?-factor. The reduced rate of curing when ethanol
was present might have been due to ethanol causing a stress
response, thus increasing the abundance of Hsp104 and corre-
spondingly decreasing the efficacy of Gdn. Because we did this
experiment with cells expressing native Sup35p, our observation
that cell division was not the determining factor in curing [PSI?]
was not due to our use of the NGMC fusion.
Although the experiments with ?-factor and farnesol may
provide the best evidence that cell division is not required for
curing [PSI?], it is interesting to note that, before it was even
known that [PSI?] was a prion, it was found that subjecting yeast
to osmotic shock converted the cells from [PSI?] to [psi?]
without cell division occurring (23, 29). In addition, Ness et al.
(15) observed that when Gdn curing of [PSI?] was done under
conditions of limiting glucose, which greatly reduces cell divi-
sion, it caused a marked decrease in sedimentable Sup35p. They
proposed that this might be due to the small amount of cell
divisionthatstilloccurred,butinlightofourresultswith?-factor
and farnesol it seems possible that it was due to dissolution of
Sup35p aggregates independently of cell division.
It has been proposed that the number of seeds in [PSI?] cells
can be estimated by determining the number of cell divisions
required to convert the cells to [psi?] (25). However, our
observation that cell division does not affect the time course of
[PSI?] curing strongly suggests that until a [PSI?] cell actually
becomes [psi?] the number of seeds in the cell is very large.
Otherwise, it might be expected that cell division would cause
enough dilution of the seeds to speed up the curing process, an
effect that we did not observe. Apparently, dissolution of
individual seeds within a cell has a much greater effect on the
time course of curing than the reduction in seed number caused
by cell division.
A question that arises is why Tuite and his collaborators (30)
observed a relatively small number of cells that were not cured
in colonies grown on plates containing Gdn. They proposed that
the seeds in the cell that began the colony remained in some of
the progeny after cell division diluted them, but another expla-
nation could be that cells at the center of colonies are in
stationary state, which would prevent them from being cured by
Gdn. In any case, because curing occurs without cell division, it
is not possible to estimate the number of seeds in the cells by
determining the number of cell divisions required to cure [PSI?].
From our data we conclude first that Hsp104 is involved in
preventing formation of large Sup35p aggregates, because we
initially observed an increase in aggregation after Hsp104 was
inactivated. Second, as originally suggested by Ness et al. (15),
the seeds that maintain [PSI?] do not have to be relatively large
aggregates, because the FRAP in [PSI?] cells can be as fast as
the rate observed in [psi?] cells. Of course, the seeds could be
relatively large oligomers of Sup35p because, as we noted above,
FRAP studies suggest that even Sup35p in [psi?] cells is much
larger than GFP. Finally, inactivation of Hsp104 does not cure
[PSI?] by preventing either large aggregates or small seeds from
being broken up and passed on to daughter cells because cell
division is not required to cure [PSI?]. Rather, in the absence of
both cell division and Hsp104 activity, an as yet unknown
mechanism completely rids the cells first of the large aggregates
and then the smaller seeds.
By using crude sedimentation analysis, Ness et al. (15) showed
that, after addition of Gdn, Sup35p aggregates did not appear to
depolymerize or to be proteolyzed during the first four cell
divisions. They also showed that newly synthesized Sup35p was
added to existing aggregates in the presence of Gdn. They
concluded that Gdn blocks seed replication but not aggregate
assembly and that cell division diluted out the Sup35p aggre-
gates, resulting in curing of [PSI?]. However, because we find
that cell division does not affect the time course of [PSI?] curing,
our data show, in fact, that proteolysis or net depolymerization
of the Sup35p aggregates must occur faster than formation of
new aggregates over the 40-h period in Gdn.
A clue to a possible curing mechanism comes from the
observation that, after ?6 h of Gdn treatment, when we observe
complete dissolution of the Sup35p aggregates, which are pre-
sumably bundles of Sup35p fibers, individual fibers have actually
undergone an ?7-fold increase in their length (ref. 16 and our
unpublished data). This finding suggests that it is the ability of
Hsp104 to sever the individual Sup35p fibers and keep them
relatively short that maintains the normal aggregates of Sup35p
observed in [PSI?] cells. On this basis, inhibition of Hsp104
activity first causes the individual Sup35p fibers to grow longer,
Wu et al.
PNAS ?
September 6, 2005 ?
vol. 102 ?
no. 36 ?
12793
CELL BIOLOGY