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Cell Death and Differentiation (2002) 9, 1043 ± 1045
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News and Commentary
Cellular responses to mitochondrial dysfunction: it's not
always downhill
RA Butow*,1
1
Department of Molecular Biology, University of Texas Southwestern Medical
Center, 5323 Harry Hines Blvd., Dallas, Texas TX 75390-9148, USA.
* Corresponding author: Tel: (214) 648-1465; Fax: (214) 648-1488;
E-mail: [email protected]
Cell Death and Differentiation (2002) 9, 1043 ± 1045.
doi:10.1038/sj.cdd.4401083
The role of mitochondria in the initiation of programmed cell
death (PCD) in metazoans needs no introduction; the role of
mitochondria in PCD in a unicellular organism, such as in the
budding yeast, Saccharomyces cerevisiae, is another matter.
There are now a number of reports indicating that when
heterologous proapoptotic proteins such as Bax are expressed in yeast, cell death may be determined by the
functional state of mitochondria.1 ± 4 Under other circumstances, such as PCD induced in yeast by osmotin, an
antifungal protein from tobacco, mitochondria do not appear
to play any role in the apoptotic response.5 This is not to say
that alterations in mitochondrial function in yeast have little
effect on yeast physiology above and beyond the strict
requirement for a fermentable carbon source to sustain
growth of cells with mitochondrial defects. Indeed, in yeast
cells with dysfunctional mitochondria, such as respiratory
deficiency brought about by the loss of mitochondrial DNA,
major rearrangements of carbohydrate and nitrogen metabolism take place through pathways of intracellular signaling
from mitochondria to the nucleus ± a response called
retrograde regulation. This involves changes in the expression of a subset of nuclear genes, the consequences of which
appear directed largely towards cellular accommodations to
the mitochondrial defects. The retrograde response is not
restricted to yeast cells, however, as there are now numerous
examples in other fungi and in animal cells of retrograde
signaling that does not elicit a cell death response.
The basic phenomenon of retrograde signaling was first
defined in yeast,6 and many of the molecular details of the
pathway have now been elaborated. Central to retrograde
signaling are three regulatory proteins, Rtg1p, Rtg2p and
Rtg3p. These factors are required for the expression of
some retrograde responsive genes, such as CIT2 and
DLD3, whose expression increases 10-fold or more in cells
with mitochondrial dysfunctions.7 ± 9 Rtg1p and Rtg3p are
basic helix-loop-helix/leucine zipper transcription factors
that interact as a heterodimer to activate transcription at
novel target sites called R boxes (GTCAC). The key to the
regulation of the retrograde response (see Sekito et al.10) is
that Rtg1p and Rtg3p are sequestered together in the
cytoplasm when the response is off. In this state, Rtg3p is
phosphorylated at multiple sites within the N-terminal part
of the protein. When the retrograde pathway is activated,
Rtg3p is partially dephosphorylated and translocates to the
nucleus; Rtg1p follows (probably passively) and the
proteins reassemble at R box sites to activate transcription.
Crucial to this regulation is Rtg2p. This protein contains an
N-terminal ATP binding domain similar to the sugar kinase/
actin/hsp70 superfamily, but its biochemical function is
unknown. Rtg2p acts a proximal sensor of mitochondrial
function, relaying mitochondrial signals to the Rtg1p/Rtg3p
complex, affecting their intracellular localization. This role
for Rtg2p is evident from the findings that in its absence,
Rtg1p and Rtg3p remain complexed in the cytoplasm
where Rtg3p is hyperphosphorylated, even in cells in which
the retrograde response would otherwise be activated.
Rtg2p does not regulate directly the intracellular
localization of Rtg1p and Rtg3p; rather, additional factors
are interposed between it and the Rtg1p/Rtg3p transcription
complex. One of these regulatory factors, Mks1p, has
appeared independently in a number of studies, but only
recently has it been recognized as an important regulator of
RTG-dependent gene expression.11 ± 13 MKS1 was originally identified as a component in Ras-cAMP signaling,14
and as a negative regulator (called lys80) of lysine
biosynthesis.15 Mks1 has also been implicated in the
regulation of the nitrogen catabolite repression (NCR)
pathway,16 which is an inductive response to low quality
nitrogen sources that allows cells to take up and to utilize
more efficiently poor nitrogen sources such as urea or
proline. In the presence of high quality nitrogen sources,
e.g., glutamine, the NCR pathway is repressed. Mks1p was
proposed to be a regulator of the NCR pathway through its
effect on Ure2p,16 a negative regulatory factor that shuts
down transcription of NCR genes by sequestering the
GATA transcription factor, Gln3p, in the cytoplasm. Of
particular interest is that Mks1p was also suggested to be
required for the formation of an inactive prion form of
Ure2p, called [URE3].17 The absence of Mks1p greatly
diminished the appearance of [URE3] cells, whereas its
overexpression stimulated [URE3] formation. Wickner18 had
established that the genetic determinant [URE3], which was
recognized some 30 years ago as a non-Mendelian trait,19
meets the essential criteria for an infectious prion. The
most recent studies show that Mks1p is likely to affect the
NCR pathway and [URE3] prion formation only indirectly
though its activity as a negative regulator of RTGdependent gene expression.11 ± 13 But as is often the case,
the devil is in the details!
The connection between the RTG pathway and nitrogen
metabolism is seen in two ways: activation of the retrograde pathway by mitochondrial dysfunction results in the
induction of a number of genes of the NCR pathway,20 and
treatment of cells with rapamycin, an inhibitor of the Tor1/2
News and Commentary
RA Butow
1044
kinases, or growth of cells on poor nitrogen sources
induces simultaneously, genes of the NCR pathway, such
as DAL5 (activated by Gln3p) and R box-containing targets
of the RTG pathway, such as CIT2 and DLD3.13,21,22 The
mechanism of these responses in terms of RTG-dependent
gene expression appears to be the same, namely, Rtg2pdependent translocation of Rtg1p and Rtg3p from the
cytoplasm to the nucleus. Discrepancies between how the
phosphorylation state Rtg3p responds to retrograde versus
NCR induction, and exactly what defines a poor nitrogen
source when comparing the RTG and NCR pathways have
not yet been resolved. Nevertheless, what is clear from
these recent studies is that Mks1p negatively regulates
RTG-dependent gene expression,11 ± 13 and that, in turn,
can affect NCR gene expression and [URE3] formation.12
In the absence of Mks1p, RTG target gene expression is
constitutive, no longer requires Rtg2p and is not repressible
by glutamate.11,12
Many of these apparently complex interrelationships are
most readily understood by recognizing that the RTG
pathway functions in glutamate homeostasis. The RTG
system monitors glutamate derived from mitochondria or
from extracellular sources via a negative feedback loop:
low levels of glutamate activate the RTG pathway and high
levels of glutamate repress it (Figure 1). Because the TCA
cycle does not operate in respiratory deficient cells, aketoglutarate production from the first three steps of the
TCA cycle must rely on anaplerotic pathways to maintain
sufficient supplies of oxaloacetate and acetyl-CoA; these
metabolites fuel those steps of the TCA cycle that produce
a-ketoglutarate, the direct precursor of glutamate. Many
genes of the NCR pathway are up-regulated when the RTG
pathway is activated, and vice versa.13,20,22 This can occur
when the NCR pathway is activated by glutamine
starvation, by rapamycin treatment or by low quality
nitrogen sources. At issue is whether the apparent
coordinate up-regulation regulation of the NCR and RTG
pathways is a general phenomenon of low quality nitrogen
sources or a function of whether the low quality nitrogen
source produces glutamate. According to Tate et al.,13 the
two can be uncoupled if the low quality nitrogen source,
when metabolized, yields repressing amounts of glutamate.
The role of the RTG pathway in the maintenance of
glutamate supplies is underscored by the fact that
expression of the genes encoding the first three steps of
the TCA cycle (catalyzed by citrate synthase, aconitase
and isocitrate dehydrogenase) leading to the synthesis of aketoglutarate are under the control of the RTG genes in
cells with reduced or compromised mitochondrial electron
transport activity.23 In cells with robust mitochondrial
respiratory activity, expression of those genes is under
HAP transcriptional control. The logic is that cellular
demands for glutamate in respiratory compromised cells
is ensured by the switch from HAP to RTG control of aketoglutarate synthesis. Strong negative regulation of those
RTG target genes by Msk1p would lead to reduction in aketoglutarate production. Because a-ketoglutarate is also
an intermediate in lysine biosynthesis, it is easy to see how
down-regulation of RTG-target gene expression would also
result in a down-regulation of lysine biosynthesis.
Cell Death and Differentiation
Figure 1 Regulation of RTG-dependent gene expression: links to TOR
signaling and [URE3] prion formation. A primary signal for mitochondrial
dysfunction is a decrease in glutamate levels due to blocks in the TCA cycle.
Through Rtg2p, Mks1p is inactivated allowing Rtg1p and Rtg3p to translocate
to the nucleus and activate target gene expression. This results in the
induction of anaplerotic pathways, increased glutamate levels and a downregulation of the RTG pathway. Increased glutamate supplies from
extracellular sources similarly down-regulates the pathway. The connections
of the RTG pathway to TOR signaling and the [URE3] prion are shown
The role of the RTG pathway in glutamate homeostasis
appears to be the key connection between the appearance
of the [URE3] prion and the retrograde pathway.12 High
levels of RTG-dependent gene expression suppress
[URE3], whereas inactivation of the pathway, for example,
in rtg mutant cells, dramatically increases [URE3] production. Glutamate, which is a potent repressor of RTGdependent gene expression, is also a potent repressor of
[URE3] formation.12 The apparent essential requirement of
Mks1p for [URE3] formation, thought to be a direct effect on
Ure2p,17 seems now likely to occur through its regulation of
the RTG pathway. As long as the Rtg1p/Rtg3p transcription
factors are intact, the absence of Mks1p will result in a
constitutively high level of RTG-target gene expression,
yielding increased glutamate (or glutamine) levels and a
suppression of [URE3] formation. Indeed, deleting MKS1
bypasses the requirement for RTG2 in RTG1/RTG3 target
gene expression. That Mks1p itself is not essential for
[URE3] formation is seen in rtg3 mks1 double mutant cells
in which the RTG pathway is inactivated but [URE3]
production is, nevertheless, high.12 Ure2p activity is
probably modulated by glutamine,24 a high quality nitrogen
News and Commentary
RA Butow
1045
source, perhaps by binding directly to the protein. Ure2p
has structural similarity to glutathione S-transferases (GST),
containing a consensus gluthathione (g-glu cys gly) binding
site, although no GST activity has been detected for
Ure2p.25,26 It is thus conceivable that Ure2p could bind
glutamate or glutamine preventing its dissociation from
Gln3p, effectively locking Ure2p in a conformation that
would suppress its conversion to [URE3].
The appearance of the [URE3] prion in cells is usually
monitored by the ability of ura2 mutants to grow on medium
containing ureidosuccinic acid (USA), a precursor of uracil.
Induction of the NCR pathway, whether by [URE3]
formation or by some other means of Ure2p inactivation,
allows cells to grow on USA medium. Genetic analysis of
the USA+ phenotype observed by Sekito et al.12 in rtg
mutant cells clearly established that the USA+ phenotype
was the result of [URE3] formation and not to some
metabolic effect unrelated to [URE3] that allowed cells to
grow on USA medium. It is important to emphasize that rtg
mutants are glutamate auxotrophs, with rtg2D mutant cells
being more leaky than rtg1 or rtg3 mutants. Thus, when
rtg2 mutant cells are cultivated on medium lacking
glutamate, glutamate starvation results in the induction of
enzymes of the NCR pathway, allowing cells to take up
USA. This is clearly a metabolic effect and is not related to
the induction of [URE3] prion formation by down-regulation
of the RTG pathway. When rtg mutant cells are grown on
medium supplemented with small amounts of glutamate, as
was done by Sekito et al.,12 the NCR pathway is repressed,
and the bulk population of cells are USA7. Importantly, the
frequency of spontaneous [URE3] formation increases
dramatically under these conditions. In other words, low
activity of the retrograde pathway initiates [URE3] formation. Because rtg mutant cells remain [URE3] after reestablishment of the RTG pathway (e.g., by introduction of
a wild-type copy of the relevant RTG gene), the RTG
pathway is not likely to be involved in the propagation of
the [URE3] prion, once formed.
How does Mks1p function to negatively regulate RTGdependent gene expression? One important clue is that
Mks1p is found in a complex with Rtg2p.12 Given the
epistatic relationship between Rtg2p and Mks1p, this
finding suggests that Rtg2p in some way modulates
Mks1p's function, inactivating it when the RTG pathway is
turned on. How that happens and how Mks1p affects the
Rtg1p/Rtg3p is not yet clear. Mks1p has been shown to be
a phosphoprotein whose phosphorylation state exactly
parallels that of Rtg3p:12 it is partially dephosphorylated
when the retrograde pathway is activated and is hyperphosphorylated in rtg2D cells. One additional clue to Mks1p's
function is that the protein contains two domains that share
sequence similarity with a putative regulatory domain in the
yeast phosphatase, Ppz1p.27 Plausibly, then, Mks1p could
modulate some phosphatase activity responsible for dephosphorylating Rtg3p when the retrograde pathway is turned
on. Clearly, additional work will be required to clarify these
regulatory steps in the retrograde pathway.
Is there a physiological rationale for the regulation of
[URE3] formation by RTG-dependent gene expression? To
answer this question, one must first ask why have prions in
yeast (or in other organisms, for that matter) in the first
place? True and Lindquist28 have argued that at least for
the yeast prion [PSI+], which affects translational termination, there may be certain circumstances in which the
epigenetic state resulting from the formation of a prion,
could be advantageous for reversible adaptation. Ure2p is
effectively a nutrient sensing device, switching on the NCR
pathway by dissociating from Gln3 when cells are faced
with a low quality nitrogen source. In the absence of other
interactions it is plausible that Ure2p would be in a
conformationally labile situation with respect to its conversion to [URE3] unless some effector (glutamate or
glutamine?) was bound to it, suppressing prion formation.
Under those conditions, operation of RTG-dependent gene
expression could supply the effector for this suppression,
allowing Ure2p to re-associate with Gln3p when a high
quality nitrogen source becomes available.
Thus we see that cells, if not exploiting mitochondrial
dysfunction, can make adjustments in the face of it through
a variety of regulatory circuits that adapt cells, sometimes
in surprising and complex ways, to the mitochondrial
disorder. Clearly PCD has its place and function. But when
a way out of mitochondrial dysfunction is needed, retrograde signaling can provide the necessary metabolic
rearrangements to make it happen.
Acknowledgements
I thank members of my laboratory for comments and criticisms. This work
was supported by grant GM22525 from the NIH and grant I-0642 from
The Robert A Welch Foundation.
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Cell Death and Differentiation