Yeast prion [PSI+] Although several dozen yeast proteins are known to behave as prions in vivo for recent advances, see refs 9-12, the prion state of the Sup35 protein – the [PSI+] deter
Trang 1Amyloid oligomers: diffuse oligomer-based transmission of yeast prions
Hideki Taguchi and Shigeko Kawai-Noma
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba, Japan
Introduction
Scrapie in sheep, bovine spongiform encephalopathy
(also called ‘mad cow’ disease) in cattle and
Creutz-feldt–Jakob disease in humans are transmissible
spong-iform encephalopathies, which are also called prion
diseases A prion is a proteinaceous infectious particle
that lacks nucleic acids, which means that it is an
infectious protein [1] In the prion, the altered
con-formers of a protein autocatalytically convert the
nor-mal structure to the altered form, which is an ordered
aggregate called amyloid Although this prion concept
was developed for the mammalian neurodegenerative
diseases in which the PrP protein participates, the
con-cept has been extended to several non-Mendelian
genetic elements in budding yeast, such as [PSI+] and
[URE3] in Saccharomyces cerevisiae [2] As yeast is
quite a tractable model eukaryote, yeast prions can provide many important insights, which are usually difficult to achieve using mammalian prions, into prion biology [3–8] In particular, the molecular mechanisms
by which the prion proteins are propagated and trans-mitted have been unraveled in the yeast prion model This review provides an overview on the transmissible entities of prion proteins in yeast prion [PSI+]
Yeast prion [PSI+]
Although several dozen yeast proteins are known to behave as prions in vivo (for recent advances, see refs 9-12), the prion state of the Sup35 protein – the [PSI+] determinant – is the best-characterized prion
Keywords
amyloid; fluorescence correlation
spectroscopy; prion; Sup35; yeast prion
Correspondence
H Taguchi, Department of Medical Genome
Sciences, Graduate School of Frontier
Sciences, University of Tokyo, FSB401,
5-1-5 Kashiwanoha, Kashiwa, Chiba, 277-8562,
Japan
Fax: +81 4 7136 3644
Tel: +81 4 7136 3644
E-mail: taguchi@k.u-tokyo.ac.jp
(Received 4 September 2009, revised 24
November 2009, accepted 25 November
2010)
doi:10.1111/j.1742-4658.2010.07569.x
Prions are infectious proteins, in which self-propagating amyloid conforma-tions of proteins are transmitted The budding yeast Saccharomyces cerevi-siae, one of the best-studied model eukaryotes, also has prions, and thus provides a tractable model system with which to understand the mechanisms
of prion phenomena The yeast prions are protein-based heritable elements, such as [PSI+], in which aggregates of prion proteins are transmitted to daughter cells in a non-Mendelian manner Although the genetic approaches preceded the yeast prion studies, recent investigations of the dynamic aspects of the prion proteins have unraveled the molecular mechanisms by which prions are propagated and transmitted In particular, several lines of evidence have revealed that the oligomeric species of prion proteins dispersed in the cytoplasm are critical for the transmission This review summarizes the topics on the transmissible entities of yeast prions, focusing mainly on the Sup35 protein in [PSI+]
Abbreviations
FAF, fluorescence autocorrelation function; FCS, fluorescence correlation spectroscopy; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; GuHCl, guanidine hydrochloride.
Trang 2Sup35 is an essential protein, which functions as a
translation termination factor in cooperation with its
partner, Sup45 [13] The N-terminal portion of Sup35
is a glutamine⁄ asparagine (Q ⁄ N)-rich domain, which
has a high propensity to form amyloid fibrils in vitro
[14–18] (Fig 1) The C-terminal domain is sufficient to
function as the termination factor (eRF3) and interacts
with Sup45 (eRF1) [13] The [PSI+] phenotype is a
nonsense suppression caused by the amyloid-like
aggregates of Sup35 [13] The propagation of [PSI+] is
strictly dependent on the presence of an appropriate
amount of Hsp104 [19] Impairment of Hsp104
func-tion, by either the deletion of Hsp104 [19] or the
addi-tion of millimolar concentrations of guanidine
hydrochloride (GuHCl), cures [PSI+] [20]
One of the central issues in prion biology is to
iden-tify the entity of the [PSI+] determinant Several
approaches, as described below, have been applied
over the past decade
Biochemical approaches to investigate
yeast prion aggregates
The proposal that non-Mendelian genetic elements
(such as [PSI+]) in yeast are prions has opened the
door to identify the molecular entity of prions [2] The
aggregates of Sup35 in the cells can be detected by
the centrifugation of yeast lysates [21,22] However,
the centrifugation assay cannot address the detailed
characteristics of the Sup35 aggregates, such as the
structure and size of prion aggregates in vivo
Kryndushkin et al developed an ingenious method
to characterize the prion aggregates in cells They
found that treatment of the [PSI+] lysate with 2% SDS disassembles the Sup35 prion aggregates into smaller, SDS-resistant particles (called polymers in their report), allowing analysis of their sizes on an aga-rose gel containing 0.1% SDS [23] This method is referred to as semidenaturing detergent–agarose gel electrophoresis [23] Using this method, they compared [PSI+] and [psi)] lysates and found that Sup35 was almost always in the oligomeric form in the [PSI+] lysate, while it was monomeric in the [psi)] lysate They concluded that the SDS-resistant prion oligomers were heterogeneous in size, ranging from 700 to
4000 kDa, which should correspond to 8-50 Sup35 monomers [23]
The possibility of other interacting proteins being present in the Sup35 oligomers was tested by affinity isolation of Sup35–His6 in [PSI+] cells [24] The affin-ity isolation revealed that the SDS-resistant Sup35 oligomers are associated with Ssa1⁄ 2 proteins in a molar ratio of 0.5 Ssa1⁄ 2 per Sup35, and with other minor components, including Hsp104, Ssb1⁄ 2, Sis1, Sse1, Ydj1 and Sla2 [24] When the affinity-purified Sup35 oligomers were negatively stained and visualized
by electron microscopy, the oligomers resembled short barrels and bundles, which seemed to be composed of barrels, rather than long fibrils [24]
Genetic approaches to investigate the transmissible entity of yeast prions
As mentioned earlier, the addition of GuHCl leads to the elimination of [PSI+] through the inhibition of Hsp104 activity [20,25–29] The kinetics of prion elimi-nation exhibited a significant lag, corresponding to around four to five generations, before the gradual emergence of [psi)] [25] Based on the kinetics, Tuite and co-workers proposed that GuHCl blocks a critical step in the replication of the prion conformers [25,30] Assuming that the prion seeds are randomly segre-gated during cell division, they calculated that the number of prion seeds, which have been named
‘propagons’, in [PSI+] cells was approximately 60 [30] Sophisticated yeast genetics revealed that [PSI+] cells contain propagons to transmit and maintain the prion phenotype
Green fluorescent protein-fused Sup35 aggregates as an indicator of [PSI+] cells
The expression of Sup35 fused with green fluorescent protein (GFP) in [PSI+] cells gives rise to the forma-tion of visible spherical fluorescent aggregates, called
Fig 1 Electron micrograph of amyloid fibrils formed by yeast prion
Sup35 Negatively stained amyloid fibrils formed by the
recombi-nant NM fragment of Sup35 [18] Bar indicates 50 nm.
Trang 3foci, in the cytosol [22] (Fig 2A), although the size
and the number of foci vary, depending on the
expres-sion level and the yeast strains used Besides the
spher-ical foci, ring⁄ rod-shaped aggregates were also
observed under some conditions, such as in [PSI+]
cells treated with GuHCl [31,32] (Fig 2B) By contrast,
Sup35–GFP does not form such visible foci, and is
dif-fusely dispersed in the [psi)] cells [22] (Fig 2C) Most
of the Sup35 in the [PSI+] cell lysate exists as
high-molecular-weight pellets, and is isolated by high-speed
centrifugation [21,22] In vitro, Sup35 fragments
containing the N domain form b-sheet-rich amyloid
aggregates [14–18] Therefore, the spherical foci are
considered to be an indicator of [PSI+] because no
such foci are seen in [psi)] cells
Regarding the relationship between the visible
Sup35–GFP foci and [PSI+], Zhou et al [31]
observed the formation of Sup35 aggregates in vivo
during the de novo induction of [PSI+] by the
overex-pression of Sup35 in [psi)][PIN+] cells, where [PIN+]
is a yeast prion phenotype that requires the induction
of [PSI+] [33] Based on their detailed examination of
the appearance of the visible aggregates in mother
and daughter cells, as well as analyses of the timing
of aggregate formation, they suggested that most of
the heritable [PSI+] seeds are too small to be
visual-ized by conventional fluorescence microscopy [31]
Song et al [34] also reported that fluorescent foci
do not directly represent [PSI+], based on the
care-ful observation of visible foci in [GPSI+] cells, in
which GFP was inserted between the N and M
domains of Sup35 in the chromosomally encoded
SUP35gene
Studying the dynamics of Sup35–GFP aggregates in living cells
Prion phenomena are intrinsically dynamic processes, because prion aggregates propagate, remodel and transmit during the protein-based inheritance in yeast prions [3,8] Simple static observations of Sup35–GFP aggregates by conventional fluorescence microscopy are insufficient to investigate the dynamic aspects of the prions in the cells Recent advances using several techniques to investigate the dynamics of protein mole-cules in living cells have provided novel insights into the molecular mechanism by which the Sup35 prion aggregates are propagated and transmitted in [PSI+] cells
a) Single-cell imaging system to monitor the fate
of the Sup35–GFP foci Several genetic analyses, combined with fluorescent microscopic observations of visible Sup35–GFP foci, have suggested that the foci do not directly represent [PSI+] However, such an ensemble method does not provide direct evidence for the significance of the visi-ble foci in the transmission of [PSI+] To gain insight into the dynamics of Sup35–GFP foci in [PSI+] cells,
an on-chip single-cell cultivation system was developed
to investigate directly the dynamic properties of prion aggregates [35,36] Specifically, the fate of the visible Sup35–GFP foci in single living [PSI+] cells was directly monitored in a time-lapse manner The on-chip cultivation system, in which the medium can
be easily exchanged during cultivation, enabled us to
Fig 2 Sup35–GFP aggregates in [PSI+] cells (A) Expression of a Sup35–GFP fusion protein in a [PSI+] cell leads to the appearance of visible spherical aggregates (foci) Phase contrast (top) and fluorescent (bottom) images are shown (B) Rod-shaped or ring-shaped visible aggre-gates are formed under the conditions where Sup35NM–GFP was overexpressed in a [PSI + ] cell that was treated with 3 5 m M guanidine hydrochloride (GuHCl) (C) Expression of Sup35NM–GFP in a prion-free [psi)] cell results in diffuse fluorescence in the cytosol Bars indicate
2 lm.
Trang 4continuously observe individual growing cells for long
periods of time The expression of Sup35–GFP was
transiently induced to lead to the formation of visible
foci in the cytoplasm of [PSI+] cells [35] After
stop-ping the further induction of Sup35–GFP, by
exchang-ing the medium with one that lacks an inducer, the
fate of the fluorescent foci was monitored in real time
Individual live-cell imaging showed that the diameter
of the foci gradually decreased, and the foci eventually
disappeared [36] The disappearance of the foci was
not caused by GFP photobleaching or degradation
[36] The disappearance of visible foci of Sup35–GFP
was also reported by analyses of a microcolony assay
system, in which preformed Sup35–GFP foci became
undetectable as the cells grew [37]
The punctate foci reappeared when Sup35–GFP
was re-induced, indicating that the seeds of the foci
were not lost in the cells after the foci disappeared
The live-cell imaging showed the appearance of the
foci in the daughter cell at almost same time as when
the foci re-appeared in the mother cell, indicating
that the seeds are transmitted from the mother cell
to the daughter cell In addition, several lines of
evidence, including a nonsense suppression assay at a
single-cell level, showed that the [PSI+] phenotype
was maintained in the cells after the foci dispersed
[36] Taken together, the single-cell imaging of
Sup35–GFP foci clearly revealed that the foci
dynam-ically dispersed into a state that functions as the
seeds of the foci and causes the nonsense
suppres-sion, which is sufficient to maintain the [PSI+]
phenotype
b) Fluorescence correlation spectroscopy
The single-cell imaging described in the previous
sec-tion revealed that, after dispersion of the foci, the
[PSI+] cells have an entity that behaves as prions
The next question is, what is the prion entity left in
the cytoplasm after the foci have dispersed?
Conven-tional fluorescence microscopic observation can barely
distinguish the difference between [PSI+] cells
with-out the foci and [psi)] cells in their appearance, as
both cells have diffuse GFP fluorescence in the
cytoplasm To elucidate the physical properties of
Sup35–GFP in living [PSI+] cells without the foci,
fluorescence correlation spectroscopy (FCS) has been
applied [36]
FCS is a technique used to analyze the diffusion
properties of fluorescent molecules, by calculating the
fluorescence autocorrelation function (FAF) in a
microscopic detection volume at the femtoliter level
[38,39] FCS allows the determination of diffusion
constants, which are directly correlated with the size of the molecules, of fluctuating fluorescent molecules under equilibrium conditions The dynamic range of FCS is very wide: FCS can measure commonly accessed diffusion dynamics on a timescale from
1 ls to 1 s Because FCS is usually combined with confocal laser-scanning microscopy, we can define the detection volume at any position of interest inside
a living cell, in a non-invasive manner As the dynam-ics of prions are basically dependent on the conversion from monomers to aggregates, and vice versa, FCS is ideally suited to estimate the size of Sup35–GFP in living yeast cells
After confirming that FCS was applicable to living yeast cells, fluorescence fluctuations of Sup35–GFP in [psi)] and [PSI+] cells, with or without the foci, were measured using FCS [36] The FAFs of Sup35–GFP
in [psi)] cells were almost the same as those in cells expressing the GFP monomer alone, indicating that [psi)] cells contain mostly monomers of Sup35–GFP
By contrast, the FAF profiles in [PSI+] cells, irre-spective of the presence of foci, were shifted to the right, compared with those in [psi)] cells, indicating that the Sup35–GFP species in [PSI+] cells were much slower, and thus larger, than those in [psi)] cells These results indicate that the larger species, referred to here as diffuse oligomers, are dispersed in the cytoplasm of [PSI+] cells, regardless of the pres-ence of foci [36]
The combination of FCS with the on-chip single cul-tivation system (a time-lapse FCS system) allows mea-surements of the size of Sup35–GFP in the daughter cells immediately after the transmission from the mother [PSI+] cells [36] Autocorrelation functions of both the mother and daughter cells were measured as the [PSI+] cell with the foci was budding Strikingly, the autocorrelation function of Sup35–GFP in the daughter cell in an early budding step was almost the same as that in the mother cell, indicating that the dif-fuse oligomers are transmissible to daughter cells [36] These time-lapse FCS experiments, combined with the retention of the seeds of the foci in the daughter cells, demonstrated that the oligomeric species dispersed in the mother cells are directly transmitted to their daughter cells [36]
The single mother–daughter pair analysis using FCS was extended to investigate the effect of Hsp104 on the transmission of Sup35–GFP [32] An FCS analysis
of GuHCl-treated [PSI+] cells revealed that Sup35– GFP diffusion in the daughter cells was faster; that is, the Sup35–GFP particle was smaller than that in the mother cells under the Hsp104-inactivated conditions [32] (see below for details)
Trang 5c) Fluorescence recovery after photobleaching
As an alternative approach to analyze the protein
dynamics in living cells, the diffusion of a fluorescent
protein can be measured using a photobleaching
tech-nique known as fluorescence recovery after
photoble-aching (FRAP) [38] In this technique, fluorescent
molecules in a small region of the cell are irreversibly
photobleached by transient exposure to a laser beam,
and the subsequent recovery of fluorescence in the
photobleached region is recorded [38] The fluorescence
intensity recovers when the fluorescent molecules
dif-fuse, as the bleached molecules diffuse away and the
unbleached molecules diffuse into the irradiated
region The kinetics of the recovery provides
informa-tion about the diffusion property of the molecules:
fas-ter recovery means fasfas-ter motion, indicating the
greater diffusion constant of the molecules As the
exposure to the laser beam for the bleaching is only
transient, this method is usually not harmful to living
cells The technique usually involves the production of
a specific protein of interest fused to GFP or other
fluorescent proteins, and has been applied to the
Sup35–GFP fusion proteins in living yeast cells
[32,34,37,40–42]
A modified [PSI+] strain, in which a functional
Sup35–GFP fusion protein (referred to as NGMC)
was created by introducing GFP between the
N-termi-nal and middle domains of endogenous Sup35, was
used for the FRAP analysis to measure the diffusion
of Sup35–GFP proteins [34] The FRAP analysis
showed that the fluorescence recovery was slower in
[PSI+] cells than in [psi)] cells, indicating that the
NGMC proteins were in an aggregated form in [PSI+]
cells [34] In a subsequent study, FRAP was used to
monitor the NGMC states in GuHCl-treated [PSI+]
cells [40] The cytoplasm in the cells showed a slower
rate of recovery after 1 hour of incubation in GuHCl,
but the rate of FRAP increased after 5 h of incubation
in the GuHCl-containing medium, and became
identi-cal to the rate observed in [psi)] cells [40] In an
inde-pendent study, FRAP was also used to measure the
physical state of Sup35–GFP in the Hsp104-inactivated
[PSI+] cells, by either the Hsp104 mutant or the
Gu-HCl treatment [37] The measurements indicated that
Sup35–GFP became largely immobile, with no
recov-ery of fluorescence in the Hsp104-inactivated cells [37]
This immobility was interpreted as a cause of the
seg-regation bias of Sup35 aggregates in the
Hsp104-inacti-vated cells, eventually leading to the loss of [PSI+]
[37]
Transmission of Sup35 from mother cells to
daugh-ter cells is critical in the prion phenomena Neither
conventional FRAP nor FCS can be used to investi-gate the flux of Sup35 between mother cells and daughter cells; however, a technique based on FRAP has been developed to investigate the flux [32] In the conventional FRAP technique, fluorescent proteins in
a small region of the cell are photobleached In the modified FRAP technique, the GFP fluorescence in the whole daughter cell is photobleached to assess the flux rate from the mother cell to the daughter cell When the modified FRAP, called MD-FRAP (mother
to daughter), was conducted with the [PSI+] cells, the flux of Sup35NM–GFP in the [psi)] cells was faster than that in the [PSI+] cells, reflecting the existence of diffuse oligomers of Sup35–GFP in the [PSI+] cells The MD-FRAP in the GuHCl-treated [PSI+] cells yielded two distinct distributions of the flux rates About half of the cells transmitted the Sup35NM– GFP with a flux rate that was almost identical to that
in the [psi)] cells, suggesting that the cells were already cured By contrast, either no, or extremely slow, flux was observed in the other half of the cells, reflecting the severe impairment of the mother–daughter trans-mission in the cells [32]
Dynamic properties of the diffuse oligomers in the [PSI+] cells
After the extension of the prion concept into yeast non-Mendelian genetic elements, a variety of tech-niques, as described above, have unraveled the molecu-lar entity of yeast prions, such as the [PSI+] determinant, over the past decade Together, the data from centrifugation assays, biochemical isolation, yeast genetics, GFP fusion methods and several single-cell approaches have revealed that the transmissible entities
of [PSI+] are the oligomeric states of Sup35 within the cytoplasm Importantly, the nature of the diffuse oligo-mers is not static, but highly dynamic Although the role of protein dynamics in prion propagation was well summarized in a recent review [8], we will further dis-cuss the details of the diffuse oligomers in the prion transmission, based mainly on our recent findings
a) Sup35–GFP foci are in equilibrium with diffuse oligomers in [PSI+] cells
Analyses using the on-chip single-cell imaging and FCS revealed the dynamics of visible foci derived from Sup35–GFP The visible fluorescent foci, which are one of the indicators of the [PSI+] phenotype, are dis-persed throughout the cytoplasm as diffuse oligomers, which are sufficient to maintain the [PSI+] phenotype [36] In addition, the diffuse oligomers are transmitted
Trang 6to their daughter cells, where the foci can reappear
[32,36] Taken together, the foci are not dead-end
aggregates, but are highly dynamic species that are in
equilibrium with the diffuse oligomers
b) What is the molecular structure of the diffuse
oligomers?
What is the molecular structure of the diffuse oligomers?
The in vitro properties of recombinant Sup35 proteins,
which form amyloid fibrils with cross b-sheet structures,
led us to hypothesize that the amyloid structures of
Sup35 are critical for the propagation and transmission
of [PSI+] in vivo However, there is no direct connection
that links the in vitro amyloid fibrils with the in vivo
transmissible entities of the prions Nevertheless, several
lines of evidence support the proposal that [PSI+] cells
contain amyloid structures of Sup35 First, in vivo,
Sup35 aggregates in [PSI+] cells are stained by an
amy-loid-staining dye, thioflavin S, indicating the presence of
cross b-sheet structures in [PSI+] cells [43] Second,
amyloid fibrils prepared from recombinant Sup35 in vitro
can efficiently convert cells from [psi)] to [PSI+] after
incorporation of the in vitro fibrils into the [psi)] cells
[44,45], indirectly showing that amyloid fibrils are
prop-agated in the [PSI+]-converted cells Third, an electron
micrographic analysis of Sup35 oligomers isolated from
[PSI+] cells revealed barrels 20 nm wide and larger
structures (bundles) [24] Although the appearance of
these structures does not resemble the typical amyloid
fibrils formed in vitro, it has been pointed out that these
structures look similar to the prion oligomers made of
recombinant Sup35, when prepared in the presence of
Hsp104 plus ATP [46] Fourth, a simulation of the FCS
data on Sup35–GFP oligomers in the [PSI+] lysate,
based on a semidenaturing detergent–agarose gel
electrophoresis analysis, clearly showed that the diffuse
oligomers are not spherical, but adopt a rod shape
(C G Pack, S Kawai-Noma, et al manuscript in
prep-aration), suggesting that the diffuse oligomers are in
amyloid-like structures Finally, electron micrographic
observations of GuHCl-treated [PSI+] cells, in which
large rod-like or ring-like aggregates of Sup35–GFP
were visible, showed that the visible rod-shaped
aggre-gates are composed of bundled fibrils (S Kawai-Noma,
A Hirata et al., manuscript in preparation) The
diame-ters of the fibrils in cells closely resemble those of
amyloid fibrils formed in vitro The bundled structure is
considered to be a consequence of the impaired Hsp104
function, which results in the formation of longer fibrils
that bundle together to form the rod-shaped structure
Taken together, the diffuse oligomers in [PSI+] cells are
most likely to be fragmented amyloid fibrils
c) Balance between growth and division of the diffuse oligomers
Prion propagation involves two distinct steps: a growth phase in which the existing amyloid particles elongate
in a self-catalyzed manner; and a division (or fragmen-tation) phase in which the amyloid particles are divided for multiplication [3] Stable maintenance of the prion phenomena relies on a delicate balance between the growth and division phases, resulting in the dynamic properties of prion particles (Fig 3) The details of the growth phase in vivo are poorly understood By contrast, the division phase has been extensively investigated because trans-acting Hsp104 and other chaperones play a critical role in this pro-cess Hsp104 is a member of the AAA+ superfamily
of ATPases (ATPases associated with various cellular activities), which is not required under normal growth conditions but is critical for surviving extreme stress, such as temperatures of 50 C [47] Hsp104, with the aid of the Hsp70⁄ 40 system, breaks protein aggregates
in an ATP-dependent manner [47] Perturbation of cel-lular Hsp104 levels dramatically affects the mainte-nance of [PSI+]: overexpression, inactivation and deletion of Hsp104 cure [PSI+] [19] Although the molecular basis by which Hsp104 overexpression cures [PSI+] remains to be solved, accumulating evidence
Fig 3 Dynamics of the yeast prion Sup35 in the cell Prion propa-gation involves two distinct steps In the growth phase, pre-existing amyloid particles elongate in a self-catalyzed manner After transla-tion at the ribosomes, Sup35 monomers (green circles) are incorpo-rated into the pre-existing amyloid fibrils (red arrowheads) In the division (or fragmentation) phase, the amyloid particles are divided for multiplication Stable maintenance of the prion phenomena relies on the delicate balance between the growth and division phases, resulting in the dynamic properties of prion particles.
Trang 7has revealed a mechanism by which the inactivation of
Hsp104 cures [PSI+]
First, microcolony observations and FRAP revealed
that the Sup35 aggregates became immobile as a result
of their increased size upon Hsp104 inactivation [37]
Second, the rod-shaped Sup35–GFP aggregates were
accumulated in GuHCl-treated [PSI+] cells [31,32],
probably because of an insufficient fragmentation of
Sup35 amyloid fibrils, which was caused by impaired
Hsp104 function Third, a single mother–daughter pair
analysis using FCS in GuHCl-treated [PSI+] cells
showed that Sup35–GFP diffusion in the daughter cells
was faster, that is, the Sup35–GFP was smaller, than
that in the mother [PSI+] cells, and it eventually
reached the diffusion profiles found in [psi)] cells [32]
Finally, MD-FRAP revealed that the flux of the
dif-fuse oligomers in the GuHCl-treated [PSI+] cells was
completely inhibited [32]
Taken together, these studies indicate that the
Hsp104 inactivation causes the severe transmission bias
between mother cells and daughter cells [32,37] In
other words, inactivation of Hsp104 alters the
dynam-ics of the diffuse oligomers of Sup35 by disrupting the
delicate balance to maintain [PSI+], eventually curing
[PSI+] So far, the mechanism by which Sup35
proteins are transmitted to daughter cells is unclear
Is there an energy-dependent transmission system, such
as the actin cytoskeleton? Alternatively, simple
diffu-sion might suffice for the transmisdiffu-sion to the daughter
cells We can assume a diffusion barrier at the bud
neck, in which the transmission of large Sup35
oligo-mers or long amyloid fibrils is more or less restricted
by an unknown mechanism
d) Size-dependent transmission of the diffuse
amyloid oligomers
Recent data on the protein dynamics under the
Hsp104-perturbed conditions prompted us to propose
that the propensity of prion entity transmission to the
daughter cell partly depends on the size of the diffuse
oligomers In this model, larger aggregates are less
transmissible to the next generations, whereas smaller
oligomers are more transmissible Considering the
observation that Sup35 forms amyloid fibrils even in
cells, we depicted the size-dependency of the
transmis-sible propensity in Fig 4A, where the short amyloid
fibrils are represented as diffuse oligomers
As extreme cases, visible foci or rod⁄ ring-shaped
aggregates are barely transmissible By contrast, the
monomeric form of Sup35 is the easiest to transmit
Therefore, diffuse oligomeric states, which have
prop-erties distinct from those of the monomer in the
protein function, would be the appropriate size for the prion phenomena of protein-based inheritance
The behavior of diffuse oligomers of Sup35 in the GuHCl-treated [PSI+] cells can be explained by an impairment of the division phase by the inactivation of Hsp104 (depicted as the absence of the division phase in Fig 4B) Moreover, this scenario can be extrapolated to the size-dependent transmission of the prion aggregates, even in the presence of functional Hsp104 (Fig 4B)
A
B
Fig 4 Proposed model of size-dependent transmission of amyloid-based oligomers (A) Prion forms of Sup35 in [PSI + ] cells are composed of diffuse oligomers, which are basically amyloid fibrils fragmented in a variety of sizes The transmissible propensity is negatively correlated with the size: shorter fibrils are more trans-missible to daughter cells In extreme cases, visible spherical foci
or rod ( ⁄ ring)-shaped aggregates are barely transmissible By con-trast, the monomeric form of Sup35 is the easiest to transmit (B) Dynamics of oligomer remodeling in [PSI + ] cells are schemati-cally represented Upper cells are normal [PSI + ] cells, in which the growth (orange arrow) and division (green arrow) of amyloid-based oligomers are well balanced Smaller oligomers are preferentially transmitted to the next generations Overexpression of Sup35 often induces the formation of spherical foci, which exist in a dynamic equilibrium with diffuse oligomers Lower cells are Hsp104-inactivated [PSI + ] cells, in which the division phase is weakened (represented as the absence of the green arrow, for emphasis) Strong transmission bias in the cells eventually leads to the cure of [PSI + ] Overexpression of Sup35 in the cells often induces the formation of visible, rod-like aggregates.
Trang 8The proposal that there is a transmission bias, even in
the presence of functional Hsp104, might extend to
yeast prion ‘strains’ and other amyloid-forming protein
phenomena [PSI+] has multiple phenotypic strains,
including strong and weak [PSI+] [48–52] The fibrils
that cause the strong [PSI+] are known to be fragile,
resulting in the smaller size [51] Size-dependent
trans-mission explains why the strong phenotype is induced
by the smaller fibrils that tend to be transmitted to the
daughter In addition, a polyglutamine sequence fused
with the C-terminal domain of Sup35 forms amyloid
aggregates, but is not inherited as a prion [53,54] If the
polyglutamine aggregates were generally large, then
the preferential retention of the large aggregates in the
mother cells might result in the impaired inheritance
Regarding the transmission from mother cells to
daughter cells, the mechanism by which Sup35 proteins
are transmitted to daughter cells is unclear Is there an
energy-dependent transmission system, such as the
actin cytoskeleton? Alternatively, simple diffusion
might suffice for the transmission to the daughter cells
We can assume that a diffusion barrier exists at the
bud neck, where the transmission of large Sup35
oligo-mers is more or less restricted by an unknown
mecha-nism In fact, because the bud neck has diffusion
barriers, such as septin rings [55,56], this assumption
might be feasible
Implication to other prions
The importance of the diffuse oligomers in [PSI+]
yeast cells could be extended to other yeast prions as
well as to mammalian prions In fact, the dynamic
nat-ure of the visible foci is not restricted to Sup35
aggre-gates in [PSI+] cells Single-cell imaging revealed that
foci derived from Rnq1–GFP in [RNQ1], a yeast prion,
also disappeared during cell growth [36], suggesting
that foci derived from other prions besides those
formed by the Sup35 and Rnq1 proteins are dynamic
during their propagation In another yeast prion
[URE3], soluble forms of the [URE3] determinant Ure2
protein were linked to the [URE3] phenotype [57],
sug-gesting that the diffuse oligomers are critical for
main-taining the [URE3] prion Regarding mammalian
prions, the physiological relevance of PrP oligomers
remains to be identified However, we note that recent
studies have shown that PrP can also form soluble
oligomeric states [58–60]
Concluding remarks
Yeast prions are not toxic amyloids Instead, amyloid
forms are utilized to switch the functional state of a
protein Typically, the monomeric form is active, whereas the aggregated (amyloid) form is inactive, which is the molecular basis of the prion phenotypes
To maintain the phenotype from generation to genera-tion, that is, the protein-based inheritance, the amyloid structures must propagate For the propagation, the amyloid fibril, an ordered aggregate, has adopted a growth-and-division strategy for the protein switch, leading to the dynamic remodeling of the diffuse oligo-mers In this context, the amyloids that are used in the prion should be fragile Understanding, in greater detail, the intrinsic fragility of the amyloids and their susceptibility to trans-acting factors, such as Hsp104, will provide important insights into prion biology as well as into other amyloid-forming proteins in the cell
Acknowledgements
We thank A Kishimoto for Figure 1 This work was supported by Grants-in-Aid for Scientific Research (B) and on Priority Areas (17370034, 18031007, 19058002
to H.T.) from JSPS and MEXT, Japan
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