The well-established abilities of peroxisomes to oxidize fatty acids to acetyl-coenzyme A (CoA) (7,8) and to use it then in anaplerotic reactions to replenish tricarboxylic acid (TCA) cycle intermediates destined for mitochondria (17,18) are integrated into several longevity regulation pathways within a cell. Such integration involves dynamic communication between peroxisomes and other organelles and is governed by the mechanisms outlined here.
Role of non-esterified fatty acids and diacylglycerol
In yeast grown in glucose-containing media and in fat storage tissues of multicellular eukaryotic organisms, the non-esterified (‘free’) fatty acids that are oxidized in peroxisomes originate mainly from triacylglycerols (TAG) and steryl esters (19–21). These neutral lipids are synthesized in the endoplasmic reticulum (ER), and are then deposited in the hydrophobic core of lipid bodies (21–23). In yeast, the lipolytic conversion of neutral lipids to free fatty acids within lipid bodies and the subsequent import of these fatty acids by peroxisomes are driven by an extensive physical contact between the two organelles (19). In fact, peroxisomes are able to invade the hydrophobic core of these stores of neutral lipids through synthesis of ‘pexopodia’(19). Recently, we proposed a mechanism that links yeast longevity to lipid dynamics in peroxisomes, lipid bodies, and the ER (4,5). This mechanism may underlie the life-extending effect of caloric restriction (CR), a low-calorie diet that increases life span in various organisms and delays the onset, or reduces the incidence, of many age-related diseases in rodent models (24). In the proposed mechanism (Figure 1), short-lived non-CR yeast accumulate ethanol, a product of glucose fermentation. By repressing the synthesis of Fox1p, Fox2p and Fox3p, all of which are the core enzymes of fatty acid β-oxidation in peroxisomes (25), ethanol suppresses peroxisomal oxidation of free fatty acids that originate from TAGs synthesized in the ER and deposited within lipid bodies (5). This leads to the accumulation of electron-dense arrays of free fatty acids (called ‘gnarls’) (19) within lipid bodies, and the initiation of several negative feedback loops. By reducing lipolysis of TAGs in lipid bodies and their biosynthesis in the ER, these feedback loops promote both the accumulation of TAG in lipid bodies and the buildup of diacylglycerol and free fatty acids in the ER (5). Because of such remodeling of lipid dynamics, which is driven by the diet-dependent changes in the efficiency of peroxisomal fatty acid β-oxidation, yeast placed on a calorie-rich diet age and die prematurely. The peroxisome-modulated premature death of non-CR yeast appears to be caused by their (i) necrotic death, triggered by the inability of peroxisomes to oxidize fatty acids which amass (26,27); (ii) caspase- and mitochondria-independent lipoapoptotic death, initiated by the accumulation of free fatty acids and diacylglycerol (28); and (iii) response to a diacylglycerol-induced reorganization of the protein kinase C-dependent signal transduction network, known to affect multiple stress response- and longevity-related processes (29,30).
Figure 1. Peroxisomal fatty acid β-oxidation acts as a system controller regulating cellular aging by modulating levels of non-esterified fatty acids and diacylglycerol. In yeast, the life-extending effect of a CR diet is due, in part, to a mechanism that links longevity to lipid dynamics in peroxisomes, lipid bodies, and the ER. Please see text for details. Figure adapted from Goldberg et al.(5).
Download figure to PowerPoint
Peroxisome metabolism and the mitochondrial retrograde signaling pathway
In response to mitochondrial dysfunction, yeast cells activate the mitochondrial retrograde (RTG) signaling pathway (31,32). Controlled by a distinct set of regulatory proteins, this signaling pathway responds to defects in mitochondria by activating transcription of a cassette of nuclear genes (Figure 2). The protein products of these genes cause a specific remodeling of carbohydrate and nitrogen metabolism, activate peroxisome proliferation, promote peroxisomal fatty acid β-oxidation and anaplerotic reactions, stimulate stress responses, and enhance the stability of nuclear and mitochondrial genomes (31–34). The resulting changes in cell physiology compensate for the mitochondrial dysfunction that prompted the response (31,32,35).
Figure 2. Peroxisomal fatty acid oxidation and anaplerotic reactions drive the RTG signaling pathway of cellular aging regulation. Functionally compromised mitochondria trigger the RTG pathway in replicatively aging yeast. Gene transcription ensues, altering cell metabolism, stimulating peroxisome function, and maintaining cell viability. Please see text for details.
Download figure to PowerPoint
The RTG pathway may be triggered in yeast cells by inducing a complete loss of mitochondrial DNA (mtDNA), introducing large deletions into mtDNA, or deleting the nuclear COX4 gene encoding one of the subunits of mitochondrial cytochrome c oxidase (36–38). The resulting loss of mitochondrial respiratory chain activity blocks the TCA cycle at succinate dehydrogenase, a four-subunit component of the chain (Figure 2) (32). Consequently, the first three reactions of the cycle—all downstream of the block—cannot convert pyruvate and acetyl-CoA to α-ketoglutarate, the direct precursor of glutamate (32). To meet the demand of nitrogen supply from glutamate to most of the biosynthetic reactions, yeast cells need to replenish acetyl-CoA, oxaloacetate and α-ketoglutarate. To achieve this, they activate the RTG signaling pathway by a yet-to-be-established mechanism. A collaborative effort of multiple protein regulators in this pathway promotes relocation of the Rtg1p–Rtg3p heterodimeric transcriptional factor from the cytosol to the nucleus, where it activates transcription of RTG-target genes (Figure 2) (31,32,35). Among these are genes for the first three enzymes in the TCA cycle, cytosolic enzymes involved in oxaloacetate and acetyl-CoA biosynthesis, a mitochondrial transporter for carnitine-dependent transfer of acetyl-CoA from the cytosol and peroxisomes to mitochondria, mitochondrial transporters for citrate and α-ketoglutarate, and plasma membrane transporters for carnitine and glutamate. In addition, RTG-target genes include a number that encode proteins required for peroxisome biogenesis and function (31–34). Among these are the Pxa1p subunit of a heterodimeric peroxisomal ATP-binding cassette transporter complex required for import of long-chain fatty acids into peroxisomes; Fox1p, Fox2p and Fox3p, the core enzymes of fatty acid β-oxidation in peroxisomes; Pex11p, a major positive regulator of peroxisome proliferation; and the citrate synthase Cit2p and acetyl-carnitine synthase Cat2p—enzymes required for the conversion of acetyl-CoA, a product of fatty acid oxidation, to citrate and acetyl-carnitine, respectively (Figure 2) (33,34). By promoting fatty acid oxidation and anaplerotic reactions in peroxisomes that replenish TCA cycle intermediates to be delivered to mitochondria, these peroxisomal proteins are vital for the ability of the RTG pathway to maintain viability of yeast cells harboring dysfunctional mitochondria (31,32). In support of this notion, the signaling pathway also responds to mitochondrial respiratory deficiency (but not to the rate of mitochondrial ATP synthesis per se) by greatly increasing peroxisome size and number under conditions that normally do not induce peroxisome assembly and proliferation (33). Moreover, in yeast cells deficient in mitochondrial respiration, the RTG pathway is mandatory for growth on oleate and for oleate-induced expression of genes whose protein products play key roles in peroxisome biogenesis and function (39,40).
It should be stressed that replicatively aging yeast gradually activate the RTG signaling pathway to compensate for the age-associated decline in their mitochondrial function which is manifest by a progressive deterioration of mitochondrial respiratory chain activity, loss of mitochondrial membrane potential, and accumulation of mutations in mtDNA (38,41–43). Importantly, the extent to which the RTG pathway is activated in response to the accumulation of dysfunctional mitochondria in yeast is directly correlated with the resulting increase in their life span (31,35,38,44). Thus, by being essential for the ability of the RTG signaling pathway to maintain viability of yeast cells harboring dysfunctional mitochondria, peroxisomes play a key role in regulating yeast longevity.
Two other nutrient-status-activated signaling pathways impact the peroxisome's ability to modulate the RTG reaction. One is the target of rapamycin (TOR) pathway, which responds primarily to the quality of the nitrogen source (45). The TOR pathway adjusts the RTG response via Lst8p, a subunit of TOR Complex 1 (TORC1) and negative regulator of the mitochondrial pathway (Figure 2) (32). Of note, a potent immunosuppressive drug, rapamycin, extends the replicative and chronological lifespans of yeast by inhibiting TORC1 (46) and, as a result, activating the RTG pathway (45). In addition, the RTG response is modulated by the Ras2p/protein kinase A signaling pathway, which responds primarily to the quality of the carbon source available (45) and impacts the pathway at Mks1p (Figure 2) (32,35).
The key point here is that the ability of peroxisomes to modulate the RTG pathway is integrated into a nutritional sensing network that regulates yeast longevity.