Our data show that the AAA protein Drg1 plays an essential role in ribosome biogenesis. Drg1 binds to late preribosomal particles, which are characterized by the presence of the pre-60S maturation factors Arx1 and Alb1. These proteins join the preribosomal particle in the nucleus and escort it into the cytoplasm (17
). Our experiments show that Drg1 joins the pre-60S particle only after its export into the cytoplasm. Moreover, we found that Drg1 is absent from the late-cytoplasmic Rei1- and Lsg1-associated pre-60S particles. These results indicate that Drg1 binds to the pre-60S subunits soon after export from the nucleus and is liberated before Lsg1 or Rei1 loading.
In affinity purifications of TAP-tagged preribosomal factors, we could detect Drg1 in complexes isolated using Rlp24, Nog1, and Arx1 as baits. Rlp24 and Arx1 are known shuttling proteins (21
) and are localized in the nucleus and the cytoplasm. In contrast, Nog1 is localized in the nucleolus and the nucleoplasm (14
) and is not detected in the cytoplasm at steady state. As Drg1 joins the preribosomal particle after export from the nucleus, the presence of Drg1 in the Nog1-TAP preparation suggests that Nog1 accompanies the preribosomal particle into the cytoplasm. This ability of Nog1 to shuttle between the nuclear and cytoplasmic compartments is further supported by the mislocalization of Nog1-GFP to the cytoplasm in a drg1-ts
mutant strain grown at restrictive temperature and upon overexpression of the dominant-negative Drg1(E617Q) protein. In contrast, Nog1-GFP does not mislocalize to the cytoplasm in a rei1
Δ strain, which provides further evidence that Rei1 acts downstream of Drg1 (A. Lebreton, C. Saveanu, and M. Fromont-Racine, unpublished results).
Inactivation of Drg1 by incubation of the temperature-sensitive drg1-ts
mutant at 37°C or depletion of the protein leads to a decrease in the amounts of several shuttling pre-60S factors (i.e., Rlp24, Nog1, Arx1, and Tif6) in the nucleus and their accumulation in the cytoplasm. The effect of Drg1 on the localization of these proteins is unlikely to be due to a simple “leaking out” of the preribosomal particles from the nucleus, since the localization of other proteins involved in similar stages of pre-60S maturation, like Drs1, Nop7, or Rlp7, remained unaffected in the mutant. We and others previously showed that Nog1, Rlp24, and Tif6 are needed for 27SB pre-rRNA processing (2
). Therefore, similar or identical processing steps are affected in Nog1-, Rlp24-, and Tif6-depleted strains and in the drg1-ts
mutant. This observation suggests that the pre-rRNA processing defects of the drg1-ts
mutant arise from the depletion of these proteins from the nucleus.
Our fluorescence microscopy, polysome profile, and affinity purification experiments show that Nog1, Rlp24, Arx1, and, to a smaller extent, Tif6 remain bound to the pre-60S particle in the cytoplasm of the drg1-ts
mutant. We conclude that the activity of Drg1 is needed for the release of these proteins from the preribosomal particles. Two additional preribosome maturation factors, which had not been included in our original studies, Nug1 and Nog2, were also enriched in Arx1-TAP purifications from the drg1-ts
mutant incubated at 37°C. Since AAA-ATPases usually act on specific proteins, it is unlikely that Drg1 is directly responsible for the release of all of these proteins. We assume that Drg1 triggers the release of one of these proteins, a process which might be crucial to set free the other preribosome maturation factors. This hypothesis is supported by the observed effect on Arx1 and Tif6. Since Arx1 and Tif6 copurify with Rei1 and Rei1 binds pre-60S particles only after dissociation of Drg1, the failure to release these proteins in the drg1-ts
mutant has to be a later consequence of Drg1 inactivation. We therefore propose the order of early cytoplasmic pre-60S maturation events shown in Fig. . Soon after export of pre-60S subunits Drg1 associates with the particles and induces the release of Nog1, Rlp24, or one of the other preribosome maturation factors identified as being enriched in our TAP purification from the drg1-ts
mutant. Drg1 dissociates from the particles either upon the release of Nog1 and/or Rlp24 or shortly after, a process which might be required for loading of Rei1. In consecutive steps, Arx1 is released and recycled in a Rei1-dependent manner (15
) and Tif6 is released by the GTPase Efl1 prior to ribosomal subunit joining (29
). The fact that Arx1 and Tif6 are blocked on pre-60S particles in the drg1-ts
mutant although they are not direct substrates of Drg1 indicates that the protein performs a conversion of the pre-60S particle which is crucial for the later release of Arx1 and Tif6. This crucial step could, for example, be the release of Nog1 or Rlp24 or one of the other proteins identified in our TAP purification from the drg1-ts
mutant. Nevertheless, our results indicate a strict order of disassembly of the shuttling preribosome maturation factors in the cytoplasm. We assume that Drg1 catalyzes the first release reaction, which in turn is the prerequisite for further maturation steps.
FIG. 9. Model for Drg1 function in late stages of pre-60S biogenesis. Only factors relevant for this work are shown. Tif6, Rlp24, Nog1, and Arx1 bind to the early pre-60S particles in the nucleus and accompany them into the cytoplasm, where Drg1 associates. Drg1 (more ...)
AAA proteins are regarded as specific chaperones which change their conformations during their ATPase cycle. The generated force may apply tension to bound proteins and so allows the ATP-dependent disruption of molecular or macromolecular structures, resulting in unfolding of proteins and/or disassembly of protein complexes (11
). Our observation that the Drg1(E617Q) protein exhibits a dominant-negative effect on the release of at least Nog1 and Rlp24 shows that ATP hydrolysis in the D2 domain of Drg1 is required for the release of shuttling proteins and therefore is necessary for maturation of the 60S subunit in the cytoplasm.
Two other AAA proteins were shown to be involved in 60S subunit formation in yeast. Rea1 and Rix7 are both required for late steps in maturation of the pre-60S subunit within the nucleus (8
). The exact role of these proteins in 60S maturation is unclear, but they are proposed to function in the remodeling of the pre-60S particle, preparation for export, and/or dissociation of nonribosomal proteins. However, factors released by Rea1 or Rix7 have yet to be identified. We show here that the ATPase activity of the essential AAA-ATPase Drg1 is required to dissociate the shuttling preribosome maturation factors Rlp24 and Nog1 from pre-60S particles, which triggers the final maturation steps of the large ribosomal subunits before they enter into translation. Generally, our results support a model in which AAA proteins are required for the release of preribosomal factors from the pre-60S particle. In contrast to Rea1 and Rix7, Drg1 performs this activity in the cytoplasm. Thus, the activity of AAA-ATPases is required at different levels in the maturation pathway of the 60S ribosomal subunit, both in the nucleus and after its export into the cytoplasm.