Transcript A yeast prion provides a mechanism for genetic variation

A yeast prion provides a
mechanism for genetic
variation and phenotypic
diversity
Heather L. True & Susan L. Lindquist
Department of Molecular Genetics and Cell Biology
Howard Hughes Medical Institute
University of Chicago
Nature – 28 September 2000
A biological problem
• Individual spontaneous
genetic changes may be
deleterious
Happy, living cells
Dead cells
A biological problem
• Individual spontaneous
genetic changes may be
deleterious
• But what if several
independent changes could
occur, that would work together
to produce a beneficial new
phenotype?
Happy, living cells
+
Dead cells
+
What if there is a way to accumulate
genetic changes “behind closed doors”
and then allow them to express
together to produce a new form or
functions?
What if there is a way to accumulate
genetic changes “behind closed doors”
and then allow them to express
together to produce a new form or
functions?
A proposed mechanism
• Gene duplication is a common
event
• A duplicated gene that is
retained in an inactive
state could accumulate
variations
• Inactive genes reactivated all at
once; cell benefits from
combinatorial changes
HOW?
Sup35 = yeast translation termination factor
• forms complex with Sup45
• Sup45 recognizes STOP codon
• Sup35 facilitates release of nascent polypeptide
RNA transcript
ribosome
Sup35 Sup45
polypeptide
RNA transcript
Sup35 ([psi-])
Has a prion form
[PSI+]
produces heritable
changes in phenotype
with no changes in
nucleic acids
[PSI+] reduces fidelity of translation
termination
→ causes ribosomes to read
through stop codons
→ suppresses nonsense mutations
Sup35 has three distinct regions
N
M
C
N-terminal
region
Middle
region
C-terminal
region
C-terminal region: responsible for translation
termination; essential for viability
N-terminal & Middle regions:
• allow Sup35 to acquire stable prion conformation
• allow cell to switch between [PSI+] and [psi-] states
• can be deleted in [psi-] cells with no apparent effect
• deletion results in loss of prion, restoration of
translation termination fidelity
Diverse growth phenotypes
produced by [PSI+]
Compared growth characteristics of [PSI+]
and [psi-] cells in >150 phenotypic assays:
• fermentable & non-fermentable carbon sources
• simple & complex nitrogen sources in presence of
salts & metals
• with inhibitors of diverse cellular processes:
–
–
–
–
DNA replication
Signalling
Protein glycosylation
Microtubule dynamics
• general stress conditions
• different temperatures
Diverse growth phenotypes
produced by [PSI+]
Examined cells from seven different genetic
backgrounds
to distinguish traits acquired from [PSI+] state
from traits arising in specific genomes
Strains
74-D694
33G-D373
SL1010-1A
D1142-1A
5V-H19
10B-H49u
BSC783/4c
• used guanidine hydrochloride curing to
create stable, isogenic pairs ([PSI+] & [psi-]
phenotypic differences would be due to [PSI+]
effects on gene expression, not genetic changes
from long-term culture
• each
strain had a different intrinsic level of
[PSI+]-mediated nonsense suppression
low- to high- levels of [PSI+] nonsense suppression
activity represented in selection of strains
Diverse growth phenotypes
produced by [PSI+]
CONTROL: Examined NM deletion strains in two
genetic backgrounds
To detect phenotypes that may be due to loss of NM region
function (which may occur with prion formation due to
occlusion), in the absence of [PSI+] activity
Strains
74-D694 DNM
33G-D373 DNM
Carefully controlled
experimental procedures
• Strain pairs examined at same growth stage
– Mid-log phase cells grown in rich medium (YPD)
– Serially diluted 5X, spotted onto test plates
• Tested amino acid supplementation in parallel experiments
– No changes in growth patterns
phenotypic differences were not owing
to effects of auxotrophic markers the strains contain
– Most strains carried adenine mutations, which turn red in the absence of
adenine ([PSI+] suppressed these mutations some of the time)
• YPD control plates were periodically interspersed with test plates
– Assured consistent spotting of cells, with same densities
• Performed repeat experiments
– Same phenotypic variances were observed each time
Marked growth differences observed
between isogenic [PSI+] & [psi-]
derivatives on many test plates
Change in colony morphology
*
Enhanced growth in [psi-] derivative
*
Enhanced growth in [PSI+] derivative
* Stronger color = stronger effect
Growth scored in two ways:
1) numbers designate the highest dilution
that produced significant growth in repeat
experiments
2) letters indicate growth rates:
R = rapid; M = medium; S = slow
V = very slow; NG = no growth
Change in growth in corresponding DNM strain
Indicated by superscript:
a = growth similar to [psi-]
b = growth similar to [PSI+]
c = different from both [psi-] & [PSI+]
For stress assays:
Growth rates: H = high; M = medium; L = low
+ = slightly increased tolerance
Change in colony morphology
*
*
Enhanced growth in [psi-] derivative
Enhanced growth in [PSI+] derivative
* Stronger color = stronger effect
Growth scored in two ways:
1) numbers designate the highest dilution
that produced significant growth in repeat
experiments
2) letters indicate growth rates:
R = rapid; M = medium; S = slow
V = very slow; NG = no growth
Change in growth in corresponding DNM strain
Indicated by superscript:
For stress assays:
a = growth similar to [psi-]
b = growth similar to [PSI+]
c = different from both [psi-] & [PSI+]
Growth rates: H = high; M = medium; L = low
+ = slightly increased tolerance
•[PSI+] does not alter growth on
YPD at pH 6.8
• [PSI+] causes strain-specific
changes at pH 6.0
•[PSI+] does not alter growth on
YPD at pH 6.8
• [PSI+] causes strain-specific
changes at pH 6.0
[psi-] grew better here
[PSI+] grew better here
[PSI+] affects growth in different ways in
different genetic backgrounds
Colony morphology changes
Cells grown in YPD, spotted onto medium with
potassium acetate as sole carbon source
[PSI+] colonies
[psi-] colonies
Spontaneous appearance
of [PSI+] colony in a group
of [psi-] cells
Cells can switch from
[PSI+] to [psi-], and vice
versa
Stress tolerance
Cells spotted on
plates with ethanol
gradient 0
10%
Cells in log phase were
incubated at 37ºC for 30
min. then transferred to
50ºC for the times indicated
[PSI+] negatively affected growth of all
strain backgrounds when cells were
plated with 5mM ZnCl2
Divergent range of phenotypes
Both these strains affected
similarly by [PSI+]
Same strains affected
very differently by [PSI+]
Strong enhancement,
but only at high
concentration of metal
[PSI+] enhances growth on
one strain, inhibits on
others, under same
conditions
[PSI+] has little effect
on most strains, but
strongly affects one
Divergent range of phenotypes
• in nearly half the conditions tested, [PSI+]
had a substantial impact in growth of at least
some strains
• in > 25% of tests, the impact on the strain
was beneficial
• each strain exhibited a unique combination
of phenotypes in response to [PSI+]
Are there common biological effects of [PSI+] ?
Enhance stress tolerance?
• no significant difference between
[PSI+] &[psi-] in either ethanol
tolerance or thermotolerance
conditions
Affect all strains in the same way?
• only once: ZnCl2
• affected all six strains capable of growth
in presence of calcofluor white
Possible biological effect of
[PSI+] on zinc metabolism or
cell wall biosynthesis
NM – specific phenotypes?
[PSI+] had both enhancing and inhibiting effects on growth in the
presence of benomyl
no common biological effect
•DNM mutants grew better in the presence of benomyl than either
[PSI+] or [psi-] variants
• DNM phenotypes were mostly different from both [PSI+] & [psi-]
The NM region of Sup35 may play a role in yeast
biology in addition to its role in [PSI+] formation
CONCLUSIONS:
• [PSI+] element of S. cerevisiae provides a means to activate silent
genetic information to produce new heritable phenotypes
• as a suppressor of nonsense mutations, [PSI+] provides mechanism for readthrough of naturally-occuring STOP codons, providing potential access to genetic
variability in duplicate genes or in 3’ non-coding regions
• allows alternative heritable phenotypes to be encoded by a single genome
• provides possibility of survival in fluctuating environment
• provides a means for evolution of new traits
• Yeast cells spontaneously switch between [PSI+] & [psi-] states
• both phenotypes are available, for adaptation to changing environment