Yeast cells missing mtDNA (rho0
) display a wide variety of changes in nuclear gene expression compared to rho+
cells (Epstein et al., 2001
; Traven et al., 2001
). The activated genes encode metabolic and stress proteins destined for the mitochondrion, the cytoplasm, and the peroxisome, and they portend a realignment of metabolism that compensates for the mitochondrial dysfunction. The loss of respiratory ability in rho0
cells eliminates the tricarboxylic acid (TCA) cycle as a source of glutamate for biosynthesis, because the activity of succinate dehydrogenase is compromised. However, the first three reactions of the TCA cycle remain intact, and this part of the TCA cycle can give rise to α-ketoglutarate, the precursor of glutamate, as long as a supply of citrate is available. This citrate is provided by the activation of the glyoxylate cycle, which utilizes oxaloacetate and acetyl-coenzyme A to generate citrate, but unlike the TCA cycle retains the carbons of acetate rather than ultimately releasing them as carbon dioxide. This central feature of the metabolic adaptation in rho0
cells is surrounded by additional niceties that allow the cell to function without an active electron transport chain. The oxidative phosphorylation that is abrogated in these cells is easily supplanted by the glycolytic production of ATP. The phenotypic changes described above are together termed the retrograde response.
The key event in retrograde signaling is the translocation of the retrograde transcription factor from the cytoplasm to the nucleus (Rothermel et al., 1995
; Rothermel et al., 1997
; Sekito et al., 2000
). The retrograde transcription factor is a heterodimer of two basic helix-loop-helix/leucine zipper proteins, Rtg1 and Rtg3, which binds to the sequence GTCAC (R box; Liao and Butow, 1993
; Jia et al., 1997
). Of the two, Rtg1 is atypical for such proteins because it possesses a truncated basic domain with no apparent transcriptional activation domains. Yet, only as a heterodimer can Rtg3 bind the R box and activate transcription. A hierarchical analysis of transcription factor regulatory networks in yeast has shown that Rtg1 is in the top layer, because it is not regulated by any other transcription factor (Jothi et al., 2009
). Top layer transcription factors are comparatively abundant, long-lived, and noisy in terms of expression from cell to cell. This variability may allow at least some members of a yeast clone or population to respond to one or another environmental challenge by launching a response whose precision is maximized by the tightly regulated transcription factors in lower layers.
Translocation of Rtg1–Rtg3 requires the Rtg2 protein (Sekito et al., 2000
), which has no known homologs in higher organisms (Figure ). Rtg2 promotes the dephosphorylation of Rtg3 by binding Mks1 and preventing Mks1 from forming a complex with the 14-3-3 protein Bmh1 or Bmh2, a complex which maintains Rtg3 in a hyperphosphorylated state (Sekito et al., 2000
; Dilova et al., 2002
; Sekito et al., 2002
; Liu et al., 2003
; Dilova et al., 2004
). Partial phosphorylation of Rtg3 is necessary, however, to expose its nuclear localization signal and thus to render the Rtg1–Rtg3 capable of activating retrograde target genes. Mks1 is removed by ubiquitin-mediated degradation promoted by the ubiquitin ligase component Grr1 (Liu et al., 2005
). Thus, Grr1 is a positive regulator of the retrograde response, while Mks1 is a negative regulator.
Figure 1 Dysfunctional mitochondria trigger a retrograde response in yeast and in round worms. In yeast, a drop in mitochondrial membrane potential (ΔΨm) initiates retrograde signaling through Rtg2, by preventing the Mks1-Bmh1/2 complex from inhibiting (more ...)
Target of rapamycin complex 1 is also a negative regulator of the retrograde response. TORC1 appears to act both upstream and downstream of Rtg2 (Komeili et al., 2000
; Giannattasio et al., 2005
; Breitkreutz et al., 2010
). One of the components of TORC1 is the WD-protein Lst8, which in genetic studies was shown to act upstream and downstream of Rtg2 depending on the identity of the mutation in Lst8 which was examined (Liu et al., 2001
; Chen and Kaiser, 2003
). This coincides well with the fact that TORC1 impinges upon the retrograde response at multiple points. The regulation of the retrograde response by TORC1 ensures that it is not active when nutrients, such as glutamate, are plentiful.
Target of rapamycin (TOR
) complex 1 is subject to negative feedback from dysfunctional mitochondria, because TORC1-mediated phosphorylation of Sch9, an AGC protein kinase, is down-regulated in rho0
cells (Kawai et al., 2011
). Phosphorylated Sch9 antagonizes stress responses under the control of the Msn2–Msn4 transcription factor and promotes ribosome biogenesis (Urban et al., 2007
). It also inhibits protein kinase A activity, balancing cell growth and metabolism with stress resistance (Zhang et al., 2011
). This occurs because protein kinase A negatively regulates Msn2–Msn4 mediated stress responses and because it feedback inhibits its own activation, which likely prevents an exaggerated response to the feedback inhibition of TORC1 by dysfunctional mitochondria. Osmotic stress also reduces Sch9 phosphorylation by TORC1, but only transiently (Urban et al., 2007
). Osmotic stress is known to recruit the Rtg1–Rtg3 transcription factor (Pastor et al., 2009
). Thus, retrograde signaling responds not only to metabolic stress but to other types of stress as well.
Ras2 is a positive regulator of the retrograde response (Kirchman et al., 1999
); however, it is not clear at which point in the retrograde signaling pathway Ras2 exerts its effect. Interestingly, MKS1
was originally identified as a negative regulator of the Ras2–cAMP pathway (Matsuura and Anraku, 1993
). This, together with the effects of TORC1 on protein kinase A suggests that it is the Ras2–cAMP pathway that contributes to the retrograde response. However, this interpretation is complicated. Activation of the retrograde response extends yeast replicative lifespan, which is measured by the number of times an individual cell divides (Kirchman et al., 1999
). Ras2 also extends replicative lifespan (Sun et al., 1994
). However, it does so via a cAMP-independent pathway. Thus, it is not clear which of the Ras2 pathways impacts the retrograde response, and indeed both the cAMP-dependent and independent pathways may be involved.
Rtg2 plays multiple roles in the cell. As discussed above, it is a positive regulator of the retrograde response, by promoting dephosphorylation of Rtg3 in the cytoplasm. In addition, it has at least two other roles in the nucleus. Rtg2 is an integral component of the transcriptional co-activator SAGA-like (SLIK) complex that contains the histone acetyltransferase Gcn5 (Pray-Grant et al., 2002
). SLIK is required for the induction of the retrograde response target gene CIT2
, and it has been shown to bind to the CIT2
promoter. The other role Rtg2 plays in the nucleus is promotion of genome stability (Bhattacharyya et al., 2002
; Borghouts et al., 2004
). The mechanism by which it extends this protection is not known, except that it does not involve the participation of an intact SLIK complex (Kim et al., 2004
The retrograde signal transducer proximal to the dysfunctional mitochondrion is Rtg2 (Liu and Butow, 2006
). However, the nature of the mitochondrial signal that triggers the retrograde response has not been clear until recently. One of the candidates was the drop in membrane potential (ΔΨm
) in dysfunctional mitochondria. Manipulation of ΔΨm
genetically, irrespective of the presence or absence of mtDNA, has shown that loss of ΔΨm
is necessary and sufficient to activate the retrograde response. However, the loss of mtDNA can augment this effect (Miceli et al., 2011
). The question now becomes how this signal is read by Rtg2. A ROS scavenger does not block the signal, and it does not appear that a drop in cellular ATP levels is involved. Thus, the loss of ΔΨm
itself must be relayed to Rtg2. Even though they are not part of retrograde regulation, mitochondrial ROS somehow signal increased chronological lifespan (survival in stationary phase) in yeast cells in which TORC1 signaling is attenuated (Pan et al., 2011
). Thus, mitochondrial ROS can perform a signaling function in some instances in yeast.
There is a gradual loss of ΔΨm
as yeasts replicatively age, which occurs without loss of mtDNA, and this is accompanied by a progressive activation of the retrograde response (Lai et al., 2002
; Borghouts et al., 2004
). Thus, it appears it is loss of ΔΨm
that triggers the retrograde response during the yeast replicative lifespan. In fact, the activation of the retrograde response may allow yeasts to live as long as they do. Indeed, the greater the forced induction of the retrograde response at the beginning of their lifespans is the greater the lifespan extension (Jazwinski, 2000
). This indicates that the retrograde response is a compensatory mechanism for mitochondrial dysfunction.
There are two other pathways that signal mitochondrial dysfunction and extend replicative lifespan that have recently been described in yeast. Mitochondrial back-signaling is activated upon deletion of the AFO1
gene, which encodes a protein found in mitochondrial ribosomes, and this activation extends replicative lifespan (Heeren et al., 2009
). This requires an active TORC1 and the transcription factor Sfp1, which activates expression of cytoplasmic ribosomal proteins. This pathway is activated only in rho0
cells. However, this occurs during growth on glucose which represses the retrograde response in the yeast strain studied. The deletion of nuclear genes that encode components of the mitochondrial translation complex (MTC), which activates translation of mtDNA-encoded proteins, also extends yeast replicative lifespan in a Sir2-dependent manner (Caballero et al., 2011
). It had been known for quite some time that interruption of mitochondrial translation with erythromycin extends yeast replicative lifespan (Holbrook and Menninger, 2002
). The relationship of mitochondrial back-signaling and the MTC to the retrograde response is of interest, but it is not known at present.