Recent studies of mammalian cells suggest that ribosomal protein (r-protein) synthesis involves control at the level of translational initiation (Thomas and Hall, 1997 ). This regulation involves recognition of an unusual pyrimidine-rich sequence at the 5′ end of r-protein mRNAs, referred to as a 5′TOP (terminal oligopyrimidine), and requires phosphorylation of ribosomal protein S6 by the P70 S6 kinase (p70^s6k) (Jefferies and Thomas, 1996 ; Jefferies et al., 1997 ; Meyuhas et al., 1996 ). In serum-starved cells as well as in cells treated with the macrolide antibiotic rapamycin, S6 becomes rapidly dephosphorylated, and 5′TOP mRNAs are no longer translated. Analysis of rapamycin-treated cells reveals that the steady-state distribution of these mRNAs shifts from polyribosomes to smaller ribonucleoprotein particles of unknown composition (Jefferies et al., 1997 ; Pedersen et al., 1997 ). The existence of 5′TOP sequences in mice as well as Xenopus suggests that this mechanism is conserved among metazoans (Meyuhas et al., 1996 ).

Rapamycin combines with the immunophilin FKBP and inhibits a large molecular weight protein termed mTOR (mammalian target of rapamycin), also known as FRAP (Thomas and Hall, 1997 ). This protein has homology to a novel family of PI-3 kinase–related kinases, whose members include Mec1, Rad3, and DNA-dependent protein kinase, and is essential for p70^s6k activity (Thomas and Hall, 1997 ). The TOR pathway also controls general translational initiation via a separate branch that is independent from p70^s6k function. In this branch, mTOR is required for activation of eIF4E, the cap-binding subunit of the eIF4F complex, probably via inhibition of 4E-BP1, which is itself an inhibitor of eIF4E function (Beretta et al., 1996 ). Thus the TOR pathway controls cell growth by coupling growth signals to changes in both general translation as well as ribosome synthesis.

Two TOR genes exist in yeast, TOR1 and TOR2, and the products of both are inhibited by rapamycin (Heitman et al., 1991 ; Helliwell et al., 1994 ; Zheng et al., 1995 ). TOR2 has an additional function involved in actin cytoskeleton dynamics and polarized cell growth; however, this function is not inhibited by rapamycin, and its relationship to protein synthesis is not well understood (Schmidt et al., 1996 , 1997 ). Rapamycin has several distinct effects on haploid yeast cells that mimic starvation, including G₁ arrest, synthesis of storage carbohydrates, onset of autophagy, and entry into G₀ (Heitman et al., 1991 ; Helliwell et al., 1994 ; Zheng et al., 1995 ; Barbet et al., 1996 ; Noda and Ohsumi, 1998 ). Rapamycin can also stimulate sporulation of diploid yeast cells under appropriate conditions (Zheng and Schreiber, 1997 ). A marked decrease in translational initiation is one of the earliest detectable effects upon treating yeast cells with rapamycin (Barbet et al., 1996 ). Indeed, inhibition of translation of the mRNA encoding Cln3, a G₁ cyclin, has been shown to be involved directly in the G₁ arrest caused by rapamycin (Barbet et al., 1996 ; Polymenis and Schmidt, 1997 ). Thus, in yeast, the TOR pathway couples protein synthesis to both cell growth and cell cycle progression in response to environmental cues. Precisely how this pathway controls translational initiation in yeast is not known, however, since TOR function has not been linked directly to eIF4E activity. A clue to this regulation has been provided by the recent demonstration that eIF4G, a subunit of the cap-binding complex that interacts with eIF4E, is degraded when cells are starved or are treated with rapamycin (Berset et al., 1998 ).

Our results also indicate that control of transcription of rRNA and r-protein genes represents a branch of the TOR pathway that is distinct from its regulation of translational initiation (Figure ​(Figure10).10). This conclusion is supported by the rapid decrease in r-protein mRNA levels observed when TOR signaling is blocked by rapamycin, in which levels decline by half in <15 min. Because this rate corresponds to the measured half-lives of these mRNAs (Kim and Warner, 1983 ) and because rapamycin acts at the level of transcription (Figure ​(Figure6),6), drug treatment must result in an essentially immediate block in r-protein gene transcription. Similarly, synthesis of 35S rRNA is severely inhibited after only brief exposure of cells to the drug. These effects rank among the earliest detectable consequences of rapamycin treatment, occurring either before or in parallel with changes in protein synthesis and in advance of any physiological sign of G₁ arrest. Taken together, these results argue for direct involvement of this pathway in the transcription of genes involved in ribosome synthesis. While this manuscript was in preparation, Schultz and coworkers reported, using an independent approach, that the TOR pathway is directly involved in the regulation of RNA Pol I and Pol III activity (Zaragoza et al., 1998 ). Because these polymerases are essential for the synthesis of 35S rRNA (Pol I) and 5S rRNA as well as tRNA (Pol III), their data are in agreement with our results.

To date, relatively few components in the TOR pathway have been identified in yeast (Figure ​(Figure10).10). In addition to Tor1 and Tor2, other proteins include 1) Pph21 and Pph22, the catalytic subunits of type 2A phosphatase (PP2A), 2) Sit4, a type 2A-related phosphatase, and 3) Tap42, a protein of unknown function that interacts with each of these phosphatases to form one or more protein complexes (collectively referred to as Tap42/PPase in Figure ​Figure10)10) (Di Como and Arndt, 1996 ; Thomas and Hall, 1997 ). A mutation in TAP42 inhibits polyribosome formation, suggesting that Tap42/PPase functions upstream of translational initiation (Di Como and Arndt, 1996 ). It is likely that Tap42/PPase also functions upstream of r-protein and rRNA gene expression for the following reasons (Figure ​(Figure10).10). First, either a mutation in TAP42 or overexpression of SIT4 confers rapamycin resistance, indicating that these proteins are likely to act before any major branch point in the TOR pathway (Di Como and Arndt, 1996 ). Second, mutations in TPD3, the gene for the regulatory A subunit of PP2A, lead to defects in the transcription of genes under control of RNA Pol III (van Zyl et al., 1992 ). Third, Sit4 has been implicated in RNA Pol II activity, and furthermore, deletion of the SIT4 gene displays synthetic lethality with a tpd3 deletion (van Zyl et al., 1992 ). These latter results provide evidence of direct involvement of type 2A phosphatases in transcription. Precisely how these phosphatases and their associated subunits regulate both protein synthesis and the activity of each of the three RNA polymerases is presently unknown.

Our results are consistent with a large body of evidence indicating that r-protein synthesis is regulated primarily at the level of transcription (Woolford and Warner, 1991 ). Indeed, it has become evident in recent years that great complexity exists in the regulation of r-protein genes in response to changes in nutrient availability. For example, it has been shown that these genes display a biphasic response during nutritional upshift, in which distinct regulatory mechanisms appear to govern an initial as well as a sustained increase in transcription (Griffioen et al., 1996 ). A model derived from these studies suggests that the initial response is independent of the growth potential of the cell, requires protein kinase A, and is regulated both by the Ras-adenylate cyclase pathway and by what has been termed the fermentable growth medium–induced pathway. This latter pathway is defined at present primarily operationally, where a strong initial increase in r-protein gene expression in the absence of adenylate cyclase activity requires a rich growth medium containing a fermentable carbon source (Thevelein, 1994 ). In contrast, a sustained increase in the transcription of r-protein genes does not require protein kinase A function but depends on the continued ability of cells to grow at an accelerated rate (Griffioen et al., 1996 ). Our results demonstrate that TOR signaling is essential for both of these steps because rapamycin treatment both inhibits steady-state expression of the r-protein genes as well as prevents their induction during nutritional upshift. The failure to induce r-protein genes during nutrient upshift in the presence of rapamycin could simply reflect a requirement for TOR signaling in general r-protein gene transcription under all conditions. Alternatively, TOR may be involved directly in the regulated expression of these genes in response to changes in nutrient availability. According to this second possibility, the TOR pathway is likely to be intimately tied to other signaling pathways involved in r-protein gene expression, possibly regulating their activity or sharing one or more components and/or targets. In this regard, it is interesting to note that the Ras-adenylate cyclase pathway affects many of the same functions regulated by TOR signaling, including nutritional control of the cell cycle, synthesis of storage carbohydrates, and entry into G₀ (Thevelein, 1994 ). Furthermore, it has been demonstrated recently that BMH1 and BMH2, which encode yeast homologues of 14-3-3 proteins, are involved in the TOR pathway (Bertram et al., 1998 ). Because these proteins have been shown previously to be involved in Ras signaling, these results provide direct evidence of at least one shared component between these pathways.

At present we do not understand how loss of TOR function results in inhibition of r-protein gene transcription. We have shown this inhibition does not require ongoing protein synthesis, apparently excluding the requirement for de novo synthesis of a transcriptional repressor. We therefore favor the idea that repression involves modification of the activity of one or more factors involved in r-protein gene activation. One obvious candidate is Rap1, a DNA-binding protein that interacts with many r-protein gene promoters and is essential for r-protein gene expression (Woolford and Warner, 1991 ). Rap1 has been shown to be involved in r-protein gene activation by the Ras-adenylate cyclase pathway (Neuman-Silverberg et al., 1995 ). This factor is also involved in transcriptional silencing at the mating type loci and at telomeres (Shore, 1994 ). Recently Rap1 has been shown to be involved in repression of r-protein gene transcription in response to perturbation of the secretory pathway (Mizuta et al., 1998 ). Specifically, it has been observed that cells expressing a deletion mutation in the RAP1 gene, the rap1-17 allele, fail to repress r-protein gene transcription at the nonpermissive temperature in temperature-sensitive sec mutants. In contrast, we have found that rapamycin inhibits r-protein gene expression equally well in both wild-type and rap1-17 cells (our unpublished results). These results suggest that the TOR pathway regulates r-protein gene expression by a mechanism that is distinct from the signaling pathway that responds to secretory defects. These results do not exclude the possibility, however, that Rap1 is nevertheless important for regulation of r-protein genes by the TOR pathway. Other candidate factors include Abf1, which also controls the activity of many r-protein genes, as well as Gcr1, a protein required for the activity of both r-protein genes as well as genes involved in glycolysis (Santangelo and Tornow, 1990 ; Tornow et al., 1993 ). In support of possible involvement of Gcr1 in TOR signaling, we have observed that rapamycin also severely inhibits expression of several glycolytic genes, including ADH1, ENO1, and PGK1 (our unpublished results).

Our results presented here are in apparent contrast to what has been reported previously for mammalian cells, in which regulation of r-protein synthesis by the TOR pathway is at the level of translational initiation (Jefferies and Thomas, 1996 ; Meyuhas et al., 1996 ; Thomas and Hall, 1997 ). These differences are consistent with the fact that in yeast r-protein mRNAs lack 5′TOP sequences and phosphorylation of ribosomal protein S6 is not essential for normal cell growth (Johnson and Warner, 1987 ), features that are required for the observed translational regulation in mammalian cells. On the other hand, it has not been reported whether rapamycin affects r-protein gene transcription in mammalian cells. Thus it is conceivable that the shift in distribution of 5′TOP mRNAs from polyribosomes to lower molecular weight ribonucleoprotein particles, observed after serum starvation or upon rapamycin treatment, pertains only to previously synthesized transcripts. According to this scenario, transcriptional control of r-protein gene expression would be a common feature among all eucaryotes, whereas translational regulation, via 5′TOP sequences, would represent an additional level of complexity that is restricted to metazoans. This scenario is consistent with evidence indicating that transcription of rRNA genes by RNA Pol I is inhibited by rapamycin in mammalian cells (Mahajan, 1994 ; Leicht et al., 1996 ).