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Originally published In Press as doi:10.1074/jbc.M604986200 on August 22, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31389-31398, October 20, 2006
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The Yeast Ccr4-Not Complex Controls Ubiquitination of the Nascent-associated Polypeptide (NAC-EGD) Complex*

Olesya Panasenko, Emilie Landrieux, Marc Feuermann, Andrija Finka, Nicole Paquet, and Martine A. Collart1

From the Department of Microbiology and Molecular Medicine, University of Geneva Medical School, 1211 Geneva 4, Switzerland

Received for publication, May 24, 2006 , and in revised form, August 16, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this work, we determine that the Saccharomyces cerevisiae Ccr4-Not complex controls ubiquitination of the conserved ribosome-associated heterodimeric EGD (enhancer of Gal4p DNA binding) complex, which consists of the Egd1p and Egd2p subunits in yeast and is named NAC (nascent polypeptide-associated complex) in mammals. We show that the EGD complex subunits are ubiquitinated proteins, whose ubiquitination status is regulated during cell growth. Egd2p has a UBA domain that is not essential for interaction with Egd1p but is required for stability of Egd2p and Egd1p. Ubiquitination of Egd1p requires Not4p. Ubiquitination of Egd2p also requires Not4p, an intact Not4p RING finger domain, and all other subunits of the Ccr4-Not complex tested. In the absence of Not4p, Egd2p mislocalizes to punctuate structures. Finally, the EGD complex can be ubiquitinated in vitro by Not4p and Ubc4p, one of the E2 enzymes with which Not4p can interact. Taken together our results reveal that the EGD ribosome-associated complex is ubiquitinated in a regulated manner, and they show a new role for the Ccr4-Not complex in this ubiquitination.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The yeast Ccr4-Not is a multifunctional complex composed of nine identified subunits (NotI-5p, Ccr4p, Caf1p, Caf40p, and Caf130p). It exists in at least two forms of 1.2 and 2 MDa that are conserved across the eukaryotic kingdom (for reviews, see Refs. 1–3). The complex is involved in several different cellular pathways. First, it regulates transcription, and controls the appropriate distribution of TFIID across promoters (4). Second, Ccr4p and Caf1p are the major deadenylase in Saccharomyces cerevisiae, and play a role in mRNA degradation (5). Third, the Ccr4-Not complex controls the post-transcriptional modification and activity of the environmental stress transcription factor Msn2p (4, 6). Taken together, the studies points to both cytoplasmic and nuclear roles for the Ccr4-Not complex, and both cytoplasmic and nuclear localizations for subunits of this complex have been described (7, 8). The precise role of the Ccr4-Not complex has not been determined, but our current model is that it serves as a platform that regulates several different cellular functions in response to changes in environmental signals, such as glucose depletion (for review, see Refs. 1 and 2).

We have little knowledge about the specific function of the individual subunits. Not4p has a conserved N-terminal domain that contains an atypical C4C4 type RING finger (9), a zinc-binding domain that defines a large family of E3 ubiquitin ligases (for review, see Ref. 10). Consistent with a role of Not4p as an E3 ligase, several E2 enzymes (UBCH6, UBCH9, and UBCH5b) were isolated in a two-hybrid selection for partners of the human CNOT4 (11). CNOT4 was subsequently shown to be capable of auto-ubiquitination in vitro, and to require its RING finger to functionally complement the deletion of NOT4 in yeast (11). Yeast Ubc4p and Ubc5p are homologous to the human E2 proteins identified as partners for CNOT4, and they interact with Not4p in the two-hybrid assay (11, 12). These yeast E2 enzymes can work with many E3 enzymes and have been associated with diverse cellular functions including the stress response, the degradation of short-lived proteins, endocytosis, and trafficking of membrane proteins and finally the degradation of cotranslationally damaged proteins (13–18).

In this work, we determine that the Ccr4-Not complex contributes to the ubiquitination and regulation of the conserved enhancer of Gal4p DNA binding (EGD)2 complex, which is named nascent polypeptide-associated complex (NAC) in mammals, and consists of the Egd1p (and its Btt1p homolog expressed at much lower levels (1/100) (19)) (betaNAC) and Egd2p ({alpha}NAC) subunits in yeast. Both subunits contain similar NAC domains through which they can dimerize and Egd2p additionally contains a ubiquitin-binding domain UBA (20) at its C terminus. The Egd1p/Egd2p heterodimer is thought to act as a dynamic component of the ribosomal exit tunnel, protecting the emerging polypeptides from interaction with other cytoplasmic proteins to ensure appropriate nascent protein targeting (for review, see Ref. 22). Recently, it was found that the ribosomal protein L25 (encoded by RPL25) is the docking site for the EGD complex to ribosomes, in close proximity to the ribosome exit tunnel (23, 24). However, the EGD complex has also been associated with transcription regulation (25–29), mostly in situations of unequal expression of either EGD subunit. It could be that the EGD complex performs both a cytoplasmic and a nuclear function, but its exact function in vivo remains to be elucidated.

We show that both Egd1p and Egd2p are ubiquitinated proteins whose ubiquitination status changes during cell growth, and we find that the ubiquitination of Egd1p and Egd2p is dependent on Not4p and the Ccr4-Not complex. We also demonstrate in vitro ubiquitination of Egd2p by Not4p and its interacting E2 enzyme Ubc4p. Finally, we find that in the absence of Not4p, Egd2p mis-localizes. Our results present the first evidence for a role of Not4p in protein ubiquitination in vivo, underline the importance of the Ccr4-Not complex for this function of Not4p, and identify a substrate for this E3 ligase. Finally our results show that Not4p contributes to the appropriate localization of its substrate in vivo.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Media and Strains—All media were standard. The strains used in this work derive from MY1 (30) (Table 1). Single-step deletions and/or tagging of genes were performed by PCR according to Ref. 31. Strain MY4448 was performed by amplifying the sequence of Egd2 between nucleotides 1 and 407. Strain MY4514 was performed by amplifying NOT4 sequences harboring the point mutation 105CTT-GCT105 (Not4L35Ap) by PCR, cloning the mutant allele of NOT4 in a URA3 integrative vector, and integration of the mutant allele at the NOT4 locus to replace endogenous NOT4.


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  		TABLE 1
Yeast strains used in this study

 
DNA Constructs—pLex202-EGD1 and pJG4–5-EGD1 were created by subcloning an EcoRI-NheI fragment from pQE30-EGD1 (kind gift from Elke Deuerling) into yEPlac112 between EcoRI and XbaI, leading to pMAC518. Then, an EcoRI-SalI fragment from pMAC518 was cloned into pLex202 or pJG4–5, leading to pMAC519 and pMAC520. pLex202-EGD2 and pJG4–5-EGD2 were created by subcloning an EcoRI-SalI fragment from pQE31-EGD2 (kind gift from Elke Deuerling) into pLex202 or pJG4–5 leading to pMAC521 and pMAC520, respectively. pJG4–5-UBA and pJG4–5-EGD2{Delta}UBA were created by amplifying the UBA domain of Egd2p (nucleotides 407 to 525) and EGD2{Delta}UBA (nucleotides 1 to 408) by PCR, and cloning into pJG4–5 between BamHI and SalI, leading to pMAC550 and pMAC551. pADH-GFP-EGD2 with or without an additional histidine tag was made by cloning a MunI-SalI or EcoRI-SalI fragment from pQE31-EGD2 into a pRS414-derived plasmid expressing GFP from the ADH1 promoter (pMAC392) between the EcoRI and XhoI sites. To make pADH-GFP-EGD2{Delta}UBA we cloned the EcoRI-EcoRV EGD2 fragment from pQE31-EGD2 into pBSSK between EcoRI and HincII, and the EcoRI-XhoI fragments from this clone were subsequently cloned into pMAC392. To construct a plasmid expressing histidine-tagged Egd1p, EGD1 sequences amplified by PCR were cloned between SacI and SpeI of pMAC334 (32) leading to pMAC538.

Two-hybrid Assays—The two-hybrid assays were performed as already described in previous studies (30). beta-Galactosidase activity was revealed by overlaying transformants with 10 ml of 0.5% agar, 0.1 M sodium phosphate buffer, pH 7.0, 0.4 mg/ml X-gal and incubating the plates for 1–24 h at 30 °C. To detect expression of bait and prey, proteins were extracted from transformants grown in 2% galactose up to an A600 of 0.6 using the post-alkaline method (33).

Recombinant Protein Purification—His6-Ubc4p (from pQE-32-NOT4; Qiagen, kind gift from Klaas Mulder) and His6-Not4p (from pET-15b, Novagen) were expressed and purified as described (9). His6-Egd1p (from pQE-30-EGD1, Qiagen) and His6-Egd2p (from pQE-31-EGD2, Qiagen) were expressed separately or co-expressed (from pQE-31-EGD2-EGD1, Qiagen, kind gift from Elke Deuerling) in MH1 cells in the presence of pRARE (Novagen) and purified as described (9).

Affinity Purification—For purification of the EGD complex using the strain expressing histidine-tagged Egd1p, 180 A600 units of cells were collected in exponential phase, resuspended in 3 ml of buffer B (50 mM Tris-HCl, pH 7.0, 20 mM imidazole, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 10 mM 2-mercaptoethanol) with 50 mM NaCl (B50) and broken during 5 min at 4 °C with 2.4 ml of glass beads. After a 20-min centrifugation of 16,000 x g at 4 °C, protein extracts were incubated with 120 µl of nickel-nitrilotriacetic acid-agarose (Qiagen) during 2 h at 4 °C, washed 3 times with 1 ml of B50 buffer and 3 times with 1 ml of B100 buffer (buffer B with 100 mM NaCl). Proteins were eluted with 60 µl of 2x Laemmli sample buffer (SB) (34). 20 µl of the samples were loaded on SDS gels and further analyzed by Western blotting.

For purification of the Ccr4-Not complex, cells expressing Caf40p-GSTp from 2 liters of culture in exponential phase were broken using FastPrep in buffer A (20 mM Tris, pH 8, 100 mM potassium acetate, 5 mM MgCl2, 10% glycerol). After ultracentrifugation (100,000 x g, 1 h) the total extract (TE) was loaded on a 1 ml of GST-trap HP column, the column was washed with 5 ml of buffer A, and proteins were eluted with 5 ml of buffer A containing 20 mM reduced glutathione. The eluted proteins were loaded on a MonoQ PC 1.6/5 column and eluted with a linear gradient of potassium acetate (0–1.5 M) on buffer A. 50 µl of the fractions were collected and analyzed with SDS-PAGE and Western blot.

Stability Assay—Cycloheximide (30 µg/ml final concentration) was added to cells growing exponentially for 24 h (A600 of 0.5). Proteins were extracted by post-alkaline lysis and 5–10 µl of the samples was analyzed with SDS-PAGE and Western blot.

In Vivo Ubiquitination Assay—Cells expressing His6-ubiquitin under the control of a copper-dependent promoter were grown in medium containing 0.1 mM CuSO4 and 50 A600 units were collected at different times of growth. Cell pellets were weighed and resuspended in G-buffer (100 mM sodium Pi, pH 8.0, 10 mM Tris-HCl, 6 M guanidium chloride, 5 mM imidazole, 0.1% Triton X-100) to 50 mg/ml. 1 ml of cell suspension was disrupted with 0.6 ml of glass beads during 6 min at 4 °C and spun for 20 min at 13,000 x g. To remove guanidium chloride, 20 µl of the supernatants were diluted in 1.2 ml of water and concentrated with Strataclean resin (Stratagene) and eluted with 50 µl of Laemmli SB. 3–5 µl of TE were analyzed by Western blot with the relevant antibodies. The rest of the supernatant was incubated with 30 µl of nickel-nitrilotriacetic acid-agarose (Qiagen) for 2 h at room temperature with mild rotation. The agarose beads were washed 3 times with 0.5 ml of U-buffer (100 mM sodium Pi, pH 6.8, 10 mM Tris-HCl, 8 M urea, 0.1% Triton X-100). His6-ubiquitinated proteins were eluted with 50 µl of 2x Laemmli SB and 12–15 µl of samples were analyzed by Western blot with the relevant antibodies.

In Vitro Ubiquitination Assay—A 150-ng aliquot of yeast E1 (Calbiochem), 150 ng of His6-Ubc4p, 200 ng of His6-Not4p, 1 µg of bovine ubiquitin (Fluka), and 1 µg of Egd1p or Egd2p or 2 µg of EDG complex were used in a 20-µl reaction containing 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 2.5 mM MgCl2, 0.5 mM EDTA, 0.25 mM dithiothreitol, 2 mM ATP, 10 mM creatine phosphate, and 5 units of creatine phosphokinase (Calbiochem). In the first case, the EGD complex was co-expressed and co-purified, in the second case Egd1p and Egd2p were expressed and purified separately and then incubated for 1 h at 30 °C in an equimolar ratio, and added to the reaction. Reaction mixtures were incubated at 30 °C for 3 h and stopped with addition of 8 µl of 4x Laemmli SB.

Live Cell Imaging—Transformed yeast cells were transferred on glass coverslips and analyzed on a Zeiss 510 confocal laser scanning microscope (LSM510). Images were recorded and image projections were assembled using the software package of the LSM 510 (Z-stack in all cases was 0.37 nm). The images were exported in tiff format and processed with Adobe Photoshop.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The NAC and Ccr4-Not Complexes Interact—We first identified an interaction between the EGD and the Ccr4-Not complexes by the two-hybrid assay. We found that Egd1p could interact with all subunits of the Ccr4-Not complex in this assay (Fig. 1A). We used two different reporter systems, one for which the growth on plates lacking leucine is indicative of an interaction, but also one for which production of beta-galactosidase is indicative of an interaction. Indeed, overproduction of certain subunits of the Ccr4-Not complex, such as Not2p, reduces cell growth, and this may prevent the visualization of a two-hybrid interaction that requires cell growth. Egd1p did not interact in this assay with several well expressed preys that consisted of subdomains of Ccr4-Not complex subunits, and the expression of the various subunits of the Ccr4-Not complex as preys did not in itself lead to transcriptional activation of the reporter genes (data not shown), indicating that the two-hybrid interactions measured were specific. The LexA-Egd2p fusion protein displayed low levels of activation and thus it was difficult to test for an interaction between the Ccr4-Not complex and Egd2p in this assay (data not shown).

To analyze a possible interaction between the EGD and Ccr4-Not complexes further, we purified the EGD complex from total protein extracts of a strain that expressed Egd1p fused to 10 histidines from an episome to functionally complement the deletion of the endogenous EGD1 gene, using a nickel column. The column eluate was analyzed by Western blotting for the presence of both EGD subunits, of a ribosomal subunit L25, because Egd1p is known to be associated with ribosomes in vivo and with this subunit in particular (23), and for the presence of some of the subunits of the Ccr4-Not complex. We determined the presence of Egd1p and Egd2p as well as L25, Not4p, and Not5p, in the column eluate (Fig. 1B). We did not detect the presence of Not3p, another subunit of the Ccr4-Not complex that often tends to dissociate during purification of the Ccr4-Not complex (data not shown). None of these proteins were detected in the eluate of an extract prepared from the host cells without the plasmid. Thus, Not4p and Not5p from a total yeast extract can be retained on a column that binds the EGD complex. In addition we were able to co-precipitate Not1p and Not5p with Egd2p (data not shown).

To determine whether the EGD complex could similarly be retained on a column that binds the Ccr4-Not complex, we created a strain expressing the Caf40p subunit of the Ccr4-Not complex fused with a GST entity. To purify the Ccr4-Not complex we prepared total protein extracts from cells expressing tagged Caf40p that we loaded on a GST-trap column. Bound proteins were eluted with reduced glutathione (Fig. 1C). The eluate of the affinity column was very dilute such that Egd2p, but not the subunits of the Ccr4-Not complex, were detected by Western blot, because of the quality of the antibodies against Egd2p. The eluate from the GST-trap column was then loaded on a MonoQ ion-exchange column, and eluted by a salt gradient. We determined the presence of all subunits of the Ccr4-Not complex, as well as Egd2p, in the same fraction of the MonoQ eluate (Caf40p, Not4p, and Egd2p are shown on Fig. 1C). Thus, Egd2p was co-purified through affinity and ion-exchange chromatography with the Ccr4-Not complex.

Not4p Is Required for Ubiquitination of Egd1p and Egd2p—The experiments so far have demonstrated that the EGD complex interacts with the Ccr4-Not complex, which contains a potential E3 ligase subunit. We thus investigated whether Egd1p and Egd2p might be ubiquitinated proteins, and tested this in cells growing in high and low glucose, because the Ccr4-Not complex has been shown to play a role under both growth conditions. To undertake this study, we created a strain expressing Egd1p fused to a triple HA epitope from its own locus and promoter and transformed this strain with a plasmid expressing ubiquitin fused to 6 histidines. We then prepared total protein extracts under denaturing conditions from cells growing at different times with gradually decreasing levels of glucose concentration (from exponential phase (100 mM of glucose) to glucose depletion (no glucose)), and purified the total protein extracts on a nickel column. The eluate was analyzed by Western blot for the presence of Egd1p-HA3p with antibodies against the HA epitope. As a control, we similarly grew and tested the same strain prior to transformation (data not shown). As shown on Fig. 2A, we detected ubiquitinated forms of Egd1p-HA3p, particularly when glucose was decreased in the growth medium. This ubiquitination of Egd1p is not due to fusion of a tag to Egd1p, because using recently obtained antibodies against Egd1p, we saw similar ubiquitination of endogenous Egd1p after the diauxic shift in cells expressing endogenous Egd1p transformed with histidine-tagged ubiquitin (data not shown).


Figure 1
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  		FIGURE 1.
The NAC and Ccr4-Not complexes interact. A, the EGY48 strain was transformed with plasmids expressing LexA-Egd1 and the indicated B42 fusion proteins. Expression of fusion proteins is shown on the left, growth of the transformants on minimal medium containing 2% galactose is shown in the middle, where serial 2-fold dilutions of transformants were spotted and grown for 3 days at 30 °C. Growth indicates a positive two-hybrid interaction. Expression of beta-galactosidase activity measured by overlay of X-gal on the grown transformants is shown on the right. B, total protein extracts were prepared from egd1{Delta} cells (lanes 1 and 3) or egd1{Delta} cells expressing histidine-tagged Egd1p (lanes 2 and 4). Total extracts (lanes 1 and 2) were loaded on a nickel column, bound proteins were eluted by the addition of SB (lanes 3 and 4) and analyzed by Western blot for the presence of Egd1p and Egd2p, L25, Not4p, and Not5p with specific polyclonal antibodies. The detected proteins are indicated on the right and molecular mass markers (in kDa) on the left. C, TE from cells expressing Caf40-GSTp was loaded on GST trap column and the flow-through (FT) was collected. The eluted proteins (E, a very dilute fraction) were loaded on an ion-exchange MonoQ column and eluted by a linear 0–1.5 M gradient of potassium acetate. Fractions from the MonoQ were analyzed by Western blot with antibodies against Caf40p, Not4p, and Egd2p. Relative exposure of blots analyzed in the left and right panels was optimized for visualization of the proteins when possible.

 
To investigate a possible role of the Not4p E3 ligase in Egd1p ubiquitination, we disrupted the NOT4 gene in the strain expressing tagged Egd1p. In this strain, we integrated at the NOT4 locus a mutant allele of NOT4 expressing a RING finger mutant of Not4p that can no longer interact with the Ubc4p and Ubc5p E2 enzymes.3 Both strains were transformed with the plasmid expressing histidine-tagged ubiquitin. Transformants were analyzed as before for Egd1p ubiquitination. We observed reduced levels of ubiquitinated Egd1p when NOT4 was disrupted, but no reduction when cells expressed a RING finger mutant of Not4p (Fig. 2A). The levels of Egd1p in the total extract were similar in the mutants and in the wild-type, and thus a decrease of total Egd1p cannot account for the reduced amount of ubiquitinated Egd1p observed in cells lacking Not4p. These results show that ubiquitination of Egd1p requires Not4p, but not its RING finger domain.


Figure 2
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  		FIGURE 2.
Not4p is required for ubiquitination of Egd1-HA3p and Egd2p. A, total protein extracts were prepared from cells expressing Egd1-HA3p and histidine-tagged ubiquitin from an episome, from exponential phase to glucose depletion (glucose concentration in the medium 100, 50, 0, 0 mM, the last point being 24 h after glucose depletion). They were loaded on a nickel column, and bound proteins were eluted by the addition of SB. TE (upper panel) and eluted proteins (Ni-eluate, lower panel) were analyzed by Western blot with antibodies against HA and secondary antibodies conjugated to peroxidase. Molecular weights of protein markers are indicated on the left. Strains used are MY1, MY4491, and MY4514. B, antibody was stripped from the nitrocellulose from A and blots were analyzed with antibodies against Egd2p and secondary antibodies conjugated to peroxidase. C, total protein extracts were prepared from wild-type, not2{Delta} (MY2181), not3{Delta} (MY3608), not4{Delta} (MY3593), not5{Delta} (MY1719), egd1{Delta} (MY3610), or egd2{Delta} (MY3609) cells expressing histidine-tagged ubiquitin from an episome. Cells were collected at the exponential phase and after glucose depletion. Egd2p from the TE (upper panel) and histidine-tagged ubiquitinated Egd2p (Ni-eluate, lower panel) was analyzed with antibodies against Egd2p and secondary antibodies conjugated to peroxidase. Molecular weights of protein markers are indicated on the left.

 
The same cells were analyzed for Egd2p ubiquitination. In wild-type cells, we detected ubiquitinated forms of Egd2p, particularly when glucose was decreased in growth medium (Fig. 2B). Ubiquitinated forms of Egd2p were also detected in cells lacking Not4p, but to a greatly reduced extent (Fig. 2B). Furthermore, the extent of Egd2p ubiquitination was also somewhat reduced in cells expressing a RING finger mutant form of Not4p (Fig. 2B). The levels of Egd2p in the total extract were similar in mutants and wild-type, and thus a decrease of total Egd2p cannot account for the reduced amount of ubiquitinated Egd2p observed in the mutants.

We next investigated the role of the other subunits of the Ccr4-Not complex in the ubiquitination of Egd2p, and for this we transformed each of the strains lacking one of the non-essential subunits of the Ccr4-Not complex with the plasmid expressing histidine-tagged ubiquitin. The transformants were tested as above. We were able to detect ubiquitinated forms of Egd2p in all strains, but to a lesser extent than in the wild-type cells, as shown in Fig. 2C for not2{Delta}, not3{Delta}, and not5{Delta} in addition to not4{Delta}. We additionally found that the level of ubiquitinated Egd2p was reduced in cells lacking Egd1p (Fig. 2C). As before, we confirmed that the levels of Egd2p in total extract were similar in all mutants and in the wild-type. Taken together these results indicate that expression of ubiquitinated Egd2p is dependent upon Not4p, its RING finger domain, the Ccr4-Not complex, and Egd1p.

Not4p and Ubc4p Ubiquitinate the EGD Complex in Vitro—The results so far have shown that Not4p E3 ligase function is important for ubiquitination of the EGD complex in vivo. However, it remains possible that this effect is indirect, particularly because the RING finger domain of Not4p was not important for Egd1p ubiquitination in vivo (see above). To thus definitively determine whether Not4p can directly ubiquitinate the EGD complex, we wanted to determine whether Not4p could ubiquitinate the EGD complex in vitro. Because Not4p interacts with Ubc4p and Ubc5p enzymes, we first tested whether these E2 enzymes could interact with Egd1p and Egd2p in a two-hybrid assay. This was indeed the case (Fig. 3A). We were also able to co-immunoprecipitate Myc-tagged Ubc4p (and Ubc5p) with Egd2p, consistently in cells expressing Not4p, but less consistently in cells lacking Not4p (data not shown). These finding suggest that an interaction of Ubc4p (and Ubc5p) with Egd2p occurs in vivo, but is supported in large part by Not4p. Thus, we next purified recombinant Not4p, Ubc4p, Egd1p, Egd2p, and the EGD complex, to set up an in vitro ubiquitination assay with commercial E1 enzyme and ubiquitin. Proteins from the assay were analyzed by Western blot with antibodies against Egd2p that also recognize Egd1p to a lesser extent. We were able to obtain a ubiquitinated form(s) of the EGD complex (Fig. 3, lane 14). This ubiquitination of the EGD complex required both Not4p and Ubc4p, because we revealed the presence of a slower migrating form of one of the EGD proteins only in the reaction that contained both Not4p and Ubc4p (Fig. 6, lane 14). Furthermore, this ubiquitination required an intact heterodimeric NAC complex, because we obtained no ubiquitination of Egd1p alone, and only very poor ubiquitination of Egd2p alone (visible in lane 9 of Fig. 3 upon very long exposure of the blot) or of Egd2p, when recombinant Egd1p was preincubated with recombinant Egd2p (visible in lane 19 of Fig. 3 upon very long exposure). Taken together, these experiments clearly demonstrate that Not4p and Ubc4p can directly ubiquitinate the Egd2p subunit of the EGD complex, and does so more efficiently when Egd2p is associated with Egd1p in a heterodimeric EGD complex formed co-translationally.


Figure 3
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  		FIGURE 3.
Ubiquitination of EGD complex by Not4p and Ubc4p in vitro. A, the interaction between Ubc4p, Ubc5p, and either Not4p or the EGD subunits was analyzed in a two-hybrid assay as described in the legend to Fig. 1A. B, ubiquitination reactions were performed for 3 h in the presence of ubiquitin, E1 activating enzyme, E2 conjugating enzyme (Ubc4p), E3 ligase (Not4p), Egd1p, (lanes 4–8) Egd2p, (lanes 9–13) or EGD complex (lanes 14–33), as indicated in the table. In samples 14–18, the EGD complex (Egd1/Egd2) subunits were co-expressed and co-purified, and in the samples 19–23, the Egd1p and Egd2p subunits were expressed and purified separately, and then added in combination to the reaction (Egd1 + Egd2). The Western blot was analyzed with anti-Egd1p and anti-Egd2p antibodies.

 
Stability of Egd1p Is Dependent upon Egd2p and Its UBA Domain—Our experiments so far show that both EGD subunits are ubiquitinated, and that the appropriate expression of ubiquitinated Egd2p requires Egd1p, and the ubiquitination of Egd2p by Not4p requires an EGD complex. We thus studied the relationship between the EGD subunits by analyzing the levels of Egd1p and Egd2p in cells lacking partner subunits of the EGD complex, with Western blot analysis of total protein extracts. As described before (19), we found that when one subunit was deleted the amount of the other was diminished (Fig. 4A). In addition, we found that the deletion of the Egd2p UBA domain led to the decrease in Egd1p as the deletion of Egd2p altogether. Interestingly, no Egd2{Delta}UBAp could be detected at all, suggesting that the trun-cated protein might be very unstable (Fig. 4A). The absence of detectable Egd2{Delta}UBAp was not due to the loss of the recognition of the truncated protein by our polyclonal antibodies because we created another UBA-less derivative of Egd2p with the fusion of an epitope at the N terminus (GFP-Egd2{Delta}UBAp) expressed from a strong promoter (ADH1) that could be visualized with our antibodies (see Fig. 5B).

As mentioned in the Introduction, there is a homolog of Egd1p in yeast that is expressed 100-fold less than Egd1p, called Btt1p (19). We thus wanted also to address the situation for this EGD subunit and so we also inspected the levels of Egd1p and Egd2p in cells lacking the BTT1 gene. We found a slight decrease of Egd1p and Egd2p in btt1{Delta} cell extracts compared with wild-type cell extracts (Fig. 4A), as described (23) for a decrease of Egd1p in cells lacking Btt1p.


Figure 4
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  		FIGURE 4.
Stability of Egd1p is dependent upon Egd2p and its UBA domain. A, the same amount of total protein extracts (confirmed by Ponceau staining) from wild-type, egd1{Delta} (MY3610), btt1{Delta} (MY3611), egd2{Delta} (MY3609), and egd2{Delta}UBA (MY4448) cells were analyzed by Western blotting using antibodies against Egd1p and Egd2p and secondary antibodies conjugated to peroxidase. B, wild-type (WT), egd1{Delta}, btt1{Delta}, egd2{Delta}, and egd2{Delta}UBA cells were grown to exponential phase for 24 h and then treated or not with cycloheximide (CHX, 30 µg/ml) as indicated. Cells were harvested after the indicated times and protein extracts made by rapid alkaline lysis were analyzed by Western blot using antibodies against Egd1p and Egd2p. C, the EGY48 yeast reporter strain was deleted for EGD2 (MY4417) and transformed with plasmids expressing LexA-Egd1p and B42 alone, or B42 fused with Egd1p, Egd2p, Egd2{Delta}UBAp, or finally to the UBA domain of Egd2p, as indicated. All of the conditions of growth were as described in the legend to Fig. 1A, and expression of all the derivatives was analyzed the same way and was similar (data not shown).

 
Because we saw slight decreases of Egd2p or Egd1p in egd1{Delta}, btt1{Delta}, or egd2{Delta} cells compared with wild-type cells, we checked the stability of Egd1p and Egd2p in cells in which a subunit of the EGD complex was deleted. To do this we grew cells in exponential phase, and treated cells or not with cycloheximide for given lengths of time, prepared extracts, and analyzed the levels of Egd1p and Egd2p by Western blot. The absence of Egd1p had no impact on the stability of Egd2p (Fig. 4B) and the deletion of Btt1p had no discernable effect on the stability of either Egd1p or Egd2p (Fig. 4B). In contrast, both the deletion of Egd2p, or the deletion of its UBA domain that also resulted in undetectable Egd2p, led to de-stabilization of Egd1p (Fig. 4B).

To determine whether absence of detectable truncated Egd2p and reduced stability of Egd1p in cells expressing UBA-less Egd2p could be due to the fact that the EGD proteins can no longer associate in an EGD complex, we performed a two-hybrid assay to look at the interaction between Egd1p and Egd2p, Egd1p and UBA-less Egd2p, or finally Egd1p and the UBA domain of Egd2p. We also analyzed the interaction of Egd1p with itself. The assay was performed in a reporter strain in which we deleted the endogenous EGD2 gene. Interestingly, Egd1p interacted similarly with itself, Egd2p, or UBA-less Egd2p, and no interaction between Egd1p and the UBA domain of Egd2p could be detected (Fig. 4C). Thus, instability of Egd1p in cells lacking Egd2p, or the instability of both Egd1p and UBA-less Egd2p, in cells expressing UBA-less Egd2p, cannot be simply explained by the impossibility of the EGD proteins to associate in an EGD complex.

Not4p Is Important for Appropriate Cellular Localization of Egd2p—Egd1p and Egd2p have been described to be associated with the ribosome, and to be able to localize in the nucleus under certain conditions. To evaluate whether the importance of Not4p for EGD ubiquitination might be related to a control of EGD complex localization, we decided to look at the localization of Egd1p and Egd2p in cells expressing or lacking Not4p. We created wild-type or not4{Delta} cells lacking the endogenous EGD1 or EGD2 genes and transformed them with plasmids expressing GFP fused to Egd1p and Egd2p, respectively, to complement the absence of the endogenous genes. Microscopic examination of the fluorescence in wild-type and not4{Delta} cells revealed no difference in the cytoplasmic localization of the GFP-Egd1p fusion protein (data not shown). In contrast, GFP-Egd2p was localized in the cytoplasm in wild-type cells, but additional discrete dots of intense fluorescence near the plasma membrane were visible in not4{Delta} cells (Fig. 5A). This mis-localization of GFP-Egd2p in cells lacking Not4p was intriguing, suggesting a mis-sorting of Egd2p to endocytic or secretory compartments. As mentioned above, Egd2p contains at its C terminus a ubiquitin-binding domain UBA (21), and membrane trafficking involves many ubiquitinated proteins. We thus analyzed the role of the UBA domain in the localization of GFP-Egd2p in wild-type and not4{Delta} cells. We constructed a plasmid expressing a mutant form of GFP-Egd2p that lacks the UBA domain, and transformed it into wild-type and not4{Delta} cells lacking endogenous Egd2p. Interestingly, the GFP-Egd2{Delta}UBAp localized similarly to the cytoplasm in wild-type and not4{Delta} cells (Fig. 5A). The Egd2p fusion proteins were appropriately expressed in all cases and the level of Egd1p was similar in wild-type or not4{Delta} cells expressing the same Egd2p derivative (Fig. 5B). Furthermore, Egd1p was stable in cells expressing the GFP-Egd2p fusion suggesting that the fusion protein was complementing the absence of the endogenous protein (data not shown). We also analyzed the ubiquitination of GFP-Egd2p in wild-type or not4{Delta} cells by transformation with a plasmid expressing His6-ubiquitin, and, similarly to endogenous Egd2p, we found that GFP-Egd2p was ubiquitinated, but less so in cells lacking Not4p (data not shown). Thus, Not4p is important for appropriate localization of Egd2p, and the Egd2p UBA domain contributes to mis-localization of Egd2p to punctuate structures in cells lacking Not4p.


Figure 5
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  		FIGURE 5.
Not4p is responsible for appropriate Egd2p localization. A, fluorescence in egd2{Delta} cells expressing or not Not4p (MY3609 and MY3623) and transformed with a plasmid expressing GFP-Egd2p or GFP-Egd2{Delta}UBAp as indicated, was measured in cells growing exponentially in high glucose. B, expression of the GFP fusion proteins and Egd1p, in both strains, was verified by Western blotting using extracts prepared from equal amounts of cells by post-alkaline lysis. The position of the GFP derivatives and Egd1p is indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A New Function and Substrate for the Ccr4-Not Complex—In this study, we determined that the yeast ribosome-associated heterodimeric EGD complex, named NAC in mammals, can interact with subunits of the Ccr4-Not complex in a two-hybrid or affinity assay. Furthermore, both subunits of the EGD complex, Egd1p and Egd2p, are ubiquitinated and in vivo this ubiquitination requires Not4p, its RING finger in the case of Egd2p, and the integrity of the Ccr4-Not complex. Finally, Not4p together with Ubc4p can ubiquitinate Egd2p within the EGD complex in vitro. Our work is the first description of the importance of the Ccr4-Not complex for protein ubiquitination in vivo. It identifies a substrate for this E3 ligase, and it shows that Not4p is important for the appropriate localization of its substrate in vivo. Thus, this study describes a novel function for the Ccr4-Not complex in vivo that has so far been described as a regulator of transcription and mRNA degradation.

Control of EGD Ubiquitination by the Ccr4-Not Complex in Vivo—Our results establish the importance of the Not4p E3 ligase for the ubiquitination of the EGD complex in vivo, and demonstrate a role for the Ccr4-Not complex in this function. An obvious question raised by our findings is how this control occurs. Indeed, we demonstrate that in vitro, Not4p and Ubc4p are capable of EGD ubiquitination, in the absence of other subunits of the Ccr4-Not complex. This ubiquitination of Egd2p in vitro is less extensive than that observed in vivo, but this could be because in vivo the Ccr4-Not complex provides a favorable docking platform for the EGD complex. Alternatively, or in addition, the Ccr4-Not complex might serve the function of regulating the moment at which ubiquitination of the EGD complex by Not4p occurs in vivo. Indeed, we have observed that ubiquitination of the EGD complex is regulated during cell growth, and this means that the enzymes that mediate these ubiquitination events must be regulated. The work so far on the Ccr4-Not complex has suggested that it serves as a regulatory platform that responds to environmental signals such as nutrient limitation or stress (for review, see Ref. 1). In particular several lines of evidence have shown that it is sensitive to glucose levels, as the ubiquitination of the EGD complex seems to be.

Our results establish that Not4p is not the only enzyme responsible for ubiquitination of Egd2p in vivo, because Egd2p ubiquitination is not abolished in cells lacking Not4p, and Not4p may not be involved in any direct ubiquitination of Egd1p, and because cells expressing the RING-finger mutant of Not4p show wild-type Egd1p ubiquitination. Thus, one can imagine that the nature and functional consequence of the ubiquitination events vary depending upon which enzyme is responsible for EGD ubiquitination.


Figure 6
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  		FIGURE 6.
Model for the involvement of the Ccr4-Not complex and Ubc4p/Ubc5p in the control of EGD ubiquitination. The Ccr4-Not complex and Ubc4p/Ubc5p, which interacts via Not4p, interacts with the EGD complex, and are important for ubiquitination of both Egd1p and Egd2p. In the absence of Not4p, ubiquitination of Egd1p and Egd2p is reduced. Egd2p protects Egd1p and itself from poly-ubiquitination and degradation through its UBA domain interacting with ubiquitinated Egd1p. Deletion of the UBA domain leads to destabilization of Egd2p, and like the total deletion of EGD2, leads to de-stabilization of Egd1p. On the other hand, reduction of Egd1p ubiquitination in not4{Delta} cells leads to relief of the Egd2p UBA domain binding ubiquitinated Egd1p. The released UBA domain contributes to mis-targeting of Egd2p in this case.

 
Another question that is raised by our results is the function for the ubiquitination of the EGD complex by Not4p in vivo. If the function of the EGD complex were clearly established, such a question would not be too difficult to answer. However, presently the function of the EGD complex is still nebulous. Indeed, the complex is associated with ribosomes in vivo, and several functions related to its interaction with nascent peptides have been proposed. In contrast, a lot of work in yeast and mammals has suggested that it can function as a transcriptional co-activator in the nucleus. It is still unclear whether the EGD complex might alternate between cytoplasmic and nuclear functions (for review, see Ref. 22).

In any event, our results have established that in cells lacking Not4p, Egd2p mis-localizes to punctuate endosomal like structures. Thus, it seems that at least one function provided by the Not4p-dependent ubiquitination of the EGD complex in vivo is the maintenance of the appropriate localization of Egd2p. Because we observed that the specific de-localization of Egd2p in cells lacking Not4p did not occur if the Egd2p UBA domain was removed, we suggest that Not4p might be responsible for a ubiquitination that is then recognized by the Egd2p UBA domain.

A recent study described the interaction between {alpha}NAC and {alpha}-taxilin, a protein involved in intracellular vesicle traffic, more particularly in the transport of vesicles to the plasma membrane (35). By extrapolation, one can imagine that in the absence of Not4p, Egd2p might localize aberrantly to punctuate structures, in part because of similar interactions between Egd2p and yeast taxilin homologues.

Functional Relevance of the Ubiquitination of the EGD Complex—An important aspect of our present work is also that it reveals that the EGD complex is ubiquitinated in a complex pattern that responds to the physiological condition of the cell. We have, however, not yet established the type of ubiquitination that the EGD complex undergoes, but we have defined an important role for the Egd2p UBA domain in the control of the stability of the EGD complex. We imagine that Not4p, together with Ubc4p or Ubc5p, contributes to ubiquitination of the EGD complex, and that the UBA domain of Egd2p is likely to recognize one of the ubiquitinated residues of the EGD complex. The UBA domain, probably through its interaction with a ubiquitinated residue, protects Egd2p itself, and in turn Egd1p, from degradation (see model on Fig. 6). An alternative explanation could be that in the absence of the UBA domain, Egd2p is inappropriately folded. However, we consider this unlikely due to the solved structure of an archaeal Egd2p homodimer (20), which shows that NAC dimerization and UBA domains of Egd2p are distinctly folded domains separated by a linker region. Finally, we observed that the N-terminal tagged derivatives of UBA-less Egd2p are much more stable than the UBA-less Egd2p, suggesting that degradation of UBA-less Egd2p might require an accessible N terminus.

Egd2p is stable in the absence of Egd1p in vivo, and it is also less ubiquitinated in this case, consistent with our finding that in vitro, ubiquitination of the EGD complex by Not4p requires both EGD subunits. Furthermore, Egd2p has been shown to relocalize to the nucleus in the absence of Egd1p and Btt1p (36), and it might relocate to a large extent already in cells lacking Egd1p only. All of these elements are likely to explain why it is stable under these conditions. Egd1p in contrast is responsible for binding the ribosome and is likely to remain cytoplasmic even in the absence of Egd2p.

The demonstration of the importance of the Egd2p UBA domain for the stability of the EGD complex is exciting. Indeed, whereas a number of ubiquitin-binding domains have now been described, there are still a few examples of the role of these domains in vivo. One recent example in yeast is the importance of the Rad23p UBA2 domain for the protection of Rad23p from proteasomal degradation (37). In that study, it was additionally shown that the UBA domain located C-terminal to several proteins, namely Dsk2p, Ddi1p, and Ede1p, protected a reporter protein fused at the N terminus to ubiquitin, from degradation. Our work is clearly in line with these published results, except that it additionally suggests that the Egd2p UBA domain is required not only for Egd2p stability but also for the stable expression of its interacting partner Egd1p.

Obviously an essential question is the role for the regulated ubiquitination of the EGD complex, besides its contribution to stability of the subunits. Because the EGD complex is associated with the ribosome and interacts with nascent peptides, a role for the complex in a quality control of nascent proteins that involves ubiquitination and proteasomal degradation of misfolded, translationally damaged, or inappropriately addressed nascent proteins is interesting to consider. It could also play an active role during the change of metabolism that occurs upon glucose depletion when the cell changes its program of gene expression. Exactly how ubiquitination of the EGD complex could contribute to these events is unclear, unless the complex can be targeted to the proteasome according to its ubiquitination status, and thereby deliver interacting proteins at the same time. In this regard, it is interesting to note that a recent study has shown the role of Ubc4p in the targeting of translationally damaged proteins (13). It was found that Ubc4p is rapidly detected in the proteasome following translational damage and we have found that the co-immunoprecipitation between Egd2p and both Not1p and Not5p increases under these conditions (data not shown). Thus, our findings lead us to consider the possibility that the EGD complex could play a role in this function, due to its privileged interaction with nascent peptides, and its capacity to be ubiquitinated by Not4p and Ubc4p. Understanding the role for the ubiquitination of the EGD complex certainly opens exciting new areas of investigation in the future.


   FOOTNOTES
 
* This work was supported by Grant 3100AO-100793 from the National Science Foundation and IHP/Network European Grant HPRN-CTG-2002-00261 supported by Office Fédéral de l'Education et de la Science (OFES) number 02.0017 (to M. A. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 41-22-3795476; Fax: 41-22-3795702; E-mail: martine.collart{at}medecine.unige.ch.

2 The abbreviations used are: EGD, enhancer of Gal4p DNA binding; NAC, nascent polypeptide-associated complex; X-gal, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside; SB, sample buffer; TE, total extract; GST, glutathione S-transferase; HA, hemagglutinin. Back

3 K. W. Mulder, A. Inagaki, E. Cameroni, F. Mousson, G. S. Winkler, C. de Virgilio, M. A. Collart, and H. T. Timmers, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank Bruno André for the plasmid expressing His6-ubiquitin, Elke Deuerling for antibodies against L25p and for plasmids allowing expressing of recombinant EGD subunits, Klaas Mulder for the plasmid allowing expressing recombinant Ubc4p and the plasmids expressing Ubc4p and Ubc5p fused to LexA in yeast, and Eve Lenssen for the plasmid expressing Not4L35A. We thank Françoise Stutz, Marc Timmers, and Thierry Lacombe for comments on our work, and Michel Strubin and Claudio de Virgilio for critical comments on our manuscript.



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 RESULTS
 DISCUSSION
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