a conserved family of wd-40 proteins binds to the ...a conserved family of wd-40 proteins binds to...

The Plant Cell, Vol. 9, 1595-1 606, September 1997 O 1997 American Society of Plant Physiologists

A Conserved Family of WD-40 Proteins Binds to the Retinoblastoma Protein in Both Plants and Animals

Robert A. Ach,’ Patti Taranto, and Wilhelm Gruissem2 Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-31 02

In mammalian cells, the retinoblastoma (RB) protein regulates G, progression and functions through its association with various cellular proteins. Two closely related mammalian RB binding proteins, RbAp48 and RbAp46, share sequence homology with the Msil protein of yeast. MSll is a multicopy suppressor of a mutation in the lRA1jgene involved in the Ras-cAMP pathway that regulates cellular growth. Human RbAp48 is present in protein complexes involved in histone acetylation and chromatin assembly. We report the cloning of cDNAs encoding four plant RbAp48- and Msil -like proteins: one from tomato, LeMSIf, and three from Arabidopsis. Complementation studies confirm that LeMSl7 can function as a multicopy suppressor of the yeast ira1 mutant phenotype. The LeMSI1 protein localizes to the nucleus and binds to a 65-kD protein in wild-type as well as ripening inhibitor (rin) and Neverripe (Nr) tomato fruit. LeMSll also binds to the human RB protein and the RB-like RRBl protein from maize, indicating that this interaction is conserved between plants and animals.

INTRODUCTION

In mammalian cells, the decision to irrevocably enter the cell cycle is made during the G, phase at the restriction point. One of the key regulators of the restriction point is the retinoblastoma (RB) protein (reviewed in Weinberg, 1995). RB was originally isolated as the product of a tumor suppressor gene, and its loss of function has been implicated in the formation of retinoblastoma and many other tumor types (reviewed in Bookstein and Lee, 1991). RB is a 11 O-kD nuclear phosphoprotein that becomes progressively phosphorylated by cyclin-dependent kinases during G, progress,ion (Ewen et al., 1993; Kato et al., 1993). RB appears to function by interact- ing with a number of cellular proteins (reviewed in Riley et al., 1994). Perhaps the best characterized of these interactions is with the E2F family of transcription factors (Lees et al., 1993), whose binding sites are found in a number of genes that are necessary for cellular proliferation and DNA synthesis (reviewed in LaThangue, 1994; Muller, 1995). Binding of E2F by RB represses transcription of E2F-regulated genes (Weintraub et al., 1995). The phosphotylation of RB during G, progression blocks this binding to E2F, thus allowing transcriptional activation (Chellappan et al., 1991).

In addition to E2F, RB binds a number of other cellular proteins, including D cyclins (Dowdy et al., 1993), RbAp48 (Qian et al., 1993), and RbAp46 (Qian et al., 1995). RbAp48

’ Current address: Hewlett-Packard Laboratories, 3500 Deer Creek Road, Palo Alto, CA 94304. To whom correspondence should be addressed. E-mail gruissem@

nature.berkeley.edu; fax 51 0-642-4995.

and RbAp46 are human nuclear proteins that are 90% identical to each other and share 30% identity with the yeast Msil protein (Ruggieri et al., 1989). MSl7 (for multicopy suppressor of ira7) is thought to be involved in the negative regulation of the yeast Ras-cAMP signaling pathway controlling cellular growth, because overexpression of the MSl7 gene suppresses the heat shock-sensitive phenotype of ira7 and r a ~ 2 ~ ~ ~ ~ ~ mutants (Ruggieri et al., 1989). The lRA7 gene (for inhibitory regulator of Ras-cAMP; Tanaka et al., 1989) encodes a yeast Ras-GTPase activating protein, whereas r a ~ 2 ~ ~ ~ ~ ~ encodes a constitutively active mutant Ras protein. Both mutants have reduced Ras-GTPase activity (Tanaka et al., 1989; Broach and Deschenes, 1990) as well as in- creased cAMP levels that are reduced by overexpression of MSl7 (Ruggieri et al., 1989). Expression of human RbAp48 or RbAp46 from multicopy plasmids in ira7 and rasvali9 cells also suppresses the mutant phenotype (Qian et al., 1993, 1995). MSl7 was also identified as a gene that when overexpressed can suppress the inability of snf4 mutants to grow on raffinose. The Snf4 protein associates with the Snfl serine/ threonine kinase, which is required for derepression of gene expression when yeast is grown on carbon sources other than glucose. Overexpression of MSl7 suppresses the snf4 growth defects by means other than inducing invertase expression, but the mechanism of this action is unknown (Hubbard et al., 1992).

Recent evidence has implicated Msil -like proteins in the regulation of histone acetylation and deacetylation and in chromatin formation. Msil was purified as a subunit of the yeast

1596 The Plant Cell

chromatin assembly complex. Although not essential for cell viability, disruption of MSl l increases UV radiation sensitivity and reduces telomeric silencing (Kaufman et al., 1997). An Msil -related protein from yeast, Hat2, has been identified as a component of a cytoplasmic histone acetyltransferase (Parthun et al., 1996). In addition, human RbAp48 was found to be a subunit of both a histone deacetylase (Taunton et al., 1996) and a chromatin assembly factor that is involved in assembling nucleosomes during DNA replication (Verreault et al., 1996). The relationships between these activities, the binding of RbAp48 and RbAp46 to RB, and the ability of Msil to suppress the Ras pathway and Snf4 mutations in yeast have not been determined to date.

In plants, cell division occurs mostly in specialized regions known as meristems, and it is highly regulated in response to developmental and environmental cues. However, in con- trast to mammals and yeast, the mechanisms that regulate entry into the plant cell cycle and its subsequent progression have not yet been elucidated. Interestingly, cDNAs encoding an RB-like protein have recently been cloned from maize (Grafi et al., 1996; Xie et al., 1996; Ach et al., 1997), indicating that at least some of the cellular machinery regulat- ing the progression of G, in mammalian cells has been conserved in plants. Plants also contain homologs of the mammalian D cyclins (Soni et al., 1995), which are involved in the phosphorylation of mammalian RB, and at least one of these plant D-like cyclins can physically bind the maize RB- like protein (Ach et al., 1997).

We describe here the cloning and analysis of cDNAs from tomato and Arabidopsis that encode proteins homologous to the human RB binding proteins RbAp48 and RbAp46 and the related yeast Msil protein. These proteins can restore the wild-type phenotype when overexpressed in ira1 and rasVa119 yeast strains. In addition to binding a 65-kD protein in wild-type and mutant fruit, we found that the tomato protein can bind to human RB as well as to RRB1, an RB-like protein for which we cloned the gene from maize (Ach et al., 1997). These results suggest that the components of the RB regulatory system found in animal cells are more evolution- arily conserved than previously thought and that the regulation of G, progression in plant cells may have a number of similarities with its regulation in mammalian cells.

RESULTS

Tomato and Arabidopsis Genes Encode Msil-like Proteins

To identify cDNAs encoding plant homologs of human RbAp48 and RbAp46 and yeast Msil, we constructed degenerate oligonucleotides corresponding to two regions conserved between human RbAp48 and RbAp46 and the yeast Msil protein (Ruggieri et al., 1989; Qian et al., 1993).

Polymerase chain reaction (PCR) amplification using cDNA libraries from young tomato fruit and Arabidopsis leaves (Ach and Gruissem, 1994) produced products of 342 bp, and sequence analysis showed that these encoded RbAp- and Msi-like proteins. Screening of the cDNA libraries with these probes resulted in the isolation of 13 clones from tomato and seven from Arabidopsis. Sequence analysis showed that 11 of the 13 tomato clones represented RbAp- and Msi-like proteins encoded by the same gene, as did all of the Arabidopsis clones. The other two tomato clones encoded unrelated proteins (data not shown). The longest of the tomato and Arabidopsis clones, 171 6 and 1538 bp, respectively, were sequenced and named LeMSll and AtMSI7. (Note: Until the biochemical function of the LeMSll and AtMSl proteins is confirmed, we have adopted the nomenclature of the gene originally identified in yeast.)

While this work was in progress, we noted an expressed sequence tag in the dbEST database (ATTS1526) that po- tentially encodes a protein closely related to that encoded by AtMSl7. Complete sequence analysis of this 1425-bp cDNA (AfMSl2) revealed that the region encoded by one of the degenerate primers from our initial PCR differed between AtMSll and AtMSl2 by two amino acids. Therefore, we conducted another PCR using a new degenerate primer directed toward this region of AtMSl2 combined with one of our original degenerate primers. A 324-bp product was ob- tained with the Arabidopsis leaf library but not with the tomato library. A full-length clone of 1461 bp was isolated, and sequencing revealed that it was the product of a third RbAp- and Msi-like Arabidopsis gene, AtMS13.

Comparison of the predicted amino acid sequences of tomato LeMSI1, Arabidopsis AtMSI1, AtMS12, and AtMS13, and human RbAp48 and RbAp46 in Figure 1 shows that these proteins are highly conserved between plants and humans. The sequence homology is spread throughout the length of the proteins. LeMSll and AtMSIl proteins are 91 % identical, and both are -65% identical to the human RbAp48 and RbAp46 sequences. The AtMS12 and AtMS13 proteins are more closely related to one another (80% identity) than they are to AtMSI1 (55% identity). The sequence homology among the plant MSI proteins is also conserved between monocots and dicots because MSI proteins encoded by a small family of at least two genes in maize are 4 0 % identical to those in Arabidopsis and tomato (H. Feiler and W. Gruissem, unpublished data). All of the proteins from plants and humans are -30% identical to the yeast Msil protein (Ruggieri et al., 1989; Kaufman et al., 1997) and the related yeast Hat2 protein (Parthun et al., 1996).

All four of the plant MSI proteins, like human RbAp48 and RbAp46 and yeast Msil and Hat2, contain seven WD-40 repeat motifs (reviewed in Neer et al., 1994). This repeat unit has been found in a number of eukaryotic regulatory proteins, such as the p subunit of trimeric G-proteins (Fong et al., 1986) and the Arabidopsis constituitive photomorpho- genic (COPI) protein involved in light-regdated signal transduction (Deng et al., 1992). The plant MSI proteins contain

Plant Retinoblastoma Binding Proteins 1597

LeMSll AtMSll AtMS12 AtMS13

Human p48 Human p46


Human p48 Human p46


Human p48 Human p46

LeMSll AtMSll AtMSI2 AtMS13

Human p48 Human p46

LeMSll AtMSll AtMSl2 AtMSl3

Human p48 Human p46

LeMSll AtMSll AtMSI2 AtMSl3

Human p48 Human p46


Human p48 Human p46

LeMSll AtMSll AtMSl2 AtMSI3

Human p48 Human p46

LeMSll AtMSll AtMSl2 AtMS13

Human p48 Human p46

Figure 1. Comparison of the Human RbAp48 and RbAp46.

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Deduced Amino Acid Sequence of Tomato LeMSll with AtMSll , AtMS12, and AtMS13 from Arabidopsis and

Dashes indicate the identity of Arabidopsis and human (Qian et al., 1993; Qian and Lee, 1995) proteins to the LeMSI1 sequence; spaces indicate gaps in homology. Arrows mark the regions corresponding to the degenerate PCR primers. Boxed amino acids show the boundaries of potential WD-40 repeats (Neer et al., 1994). The lengths of the cDNAs and the location of their coding regions are as follows: the LeMSl7 cDNA is 1716 bp, with the coding region from positions 145 to 1416; the AfMSll cDNA is 1538 bp, with the coding region from positions 76 to 1360; the AtMS12 cDNA is 1425 bp, with the coding region from positions 6 to 1250; and the AfMSW cDNA is 1461 bp, with the coding region from positions 4 to 1275. GenBank accession numbers are AF016845, AF016846, AF016847, and AF016848.

1598 The Plant Cell

seven repeats marked by the characteristic core element of(G,V,A,N,S)H-X28-(W,F)D, where X stands for any amino acid(Neer et al., 1994). Although other amino acids are con-served between repeats, the individual repeats of each MSIprotein share more homology with the corresponding repeatin the other proteins than with the other repeating unitswithin the same protein. The regions at the N and C terminiof each protein, outside of the WD-40 repeats, also sharesequence homology and are rich in acidic residues.

To determine whether LeMSH is a member of a multigenefamily in tomato, as in Arabidopsis, genomic DMA gel blotanalysis (Figure 2) was performed using a probe containingnucleotides 632 to 792 of the LeMSIt cDNA. One band wasdetected in Xhol, Hindlll, and BamHI digests, and two bandsof equal intensity were seen in EcoRI and Xbal digests.When the blot was reprobed and washed under less strin-gent conditions, no additional bands were detected. We con-clude that LeMSIl is a single-copy gene in tomato, althoughwe cannot rule out the existence of other more distantly re-lated MSI-encoding genes.

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LeMSH Is an Abundant, Ubiquitously ExpressedTranscript in Tomato

To determine the expression pattern of LeMSH in varioustissues, we performed RNA gel blot analysis using the sameLeMSH cDNA probe that was used in the DMA gel blot anal-ysis. Figure 3 shows that in all of the tissues examined, wedetected a single mRNA of 1.7 kb, corresponding to thelength of the LeMSH cDNA. LeMSH mRNA levels are high-est in developing fruit, which has a large number of rapidlydividing cells (Gillaspy et al., 1993). However, the mRNAwas present in all tissues, including tissues in such fully dif-ferentiated organs as red fruit and leaves, suggesting thatLeMSH functions in all cells.

Figure 2. Gel Blot Analysis of Genomic DNA from Tomato.

Genomic DNA (10 |xg) was digested with the indicated restrictionenzymes, fractionated in an 0.8% agarose gel, transferred to a nylonmembrane, and probed with a 32P-labeled fragment from theLeMSH coding region. DNA length markers are given at left in kilo-bases.

Fruit

LeMSH

18S rRNA

Figure 3. Expression of LeMSH mRNA in Tomato.

Samples (5 ̂ .g) of total RNA from various tomato tissues were fractionated in a 1 % agarose-formaldehyde gel, transferred to a nylon membrane,and probed with a 32P-labeled probe from the LeMSH coding region. As a control for loading and transfer, the blot was stripped and rehybrid-ized with an 18S rRNA probe (Ach and Gruissem, 1994). Lt. Cotyl., RNA from 7-day-old cotyledons grown in 12 hr of darkness and 12 hr of light;Dk. Cotyl., RNA from 7-day-old cotyledons grown in constant darkness; MG, RNA from mature green fruit.


LeMSM Localizes to the Nucleus, and Protein LevelsAre Not Correlated with Cell Division Activity

Protein gel blot analysis using an affinity-purified mouseanti-LeMSH antiserum showed that the LeMSH protein, likethe mRNA, is detectable in all organs in tomato (data notshown). To examine the distribution of LeMSII in more de-tail, we used the anti-LeMSIt antiserum to perform immu-nolocalizations in sections of young tomato fruit and floralmeristems, as shown in Figure 4. In both tissues, immunore-activity against the anti-LeMSM antiserum was detected inthe nuclei of nearly all cells (Figures 4B and 4D) but not withthe anti-LeMSH-depleted antiserum (Figures 4A and 4C).This confirms that LeMSII is a nuclear protein, as is mam-malian RbAp48 (Qian et al., 1993). The level of LeMSII stain-ing, however, does not correlate with cell division activity inthe tissues we examined. In the young developing fruit (~1cm; Figure 4B), the most intensely stained nuclei are foundin the pericarp, where cell expansion and differentiation oc-cur and cell division has mostly ceased (Gillaspy et al., 1993).Nuclei in the epidermal cells stained more lightly, althoughthese cells continued to divide until later in fruit develop-ment. However, in floral buds shown in Figure 4D, the nucleiwithin the meristems, the emerging organs, and the vasculartissue stained most strongly. Cells in these regions are rela-tively undifferentiated and rapidly dividing. There is thereforeno apparent correlation between the state of differentiationor cell division activity and the amount of LeMSI proteinwithin the cells.

LeMSII Can Suppress the Phenotype of Two RasPathway Mutations in Yeast

In yeast, the Ras-cAMP signal transduction pathway mediatescell growth in response to nutrients (Broach and Deschenes,1990). The IRA1 gene product negatively regulates this path-way by stimulating the GTPase activity of the Ras proteins(Tanaka et al., 1989). The yeast MSI1 gene was originally iso-lated in a screen for multicopy suppressors of the heat shock-sensitive phenotype of the iral mutation (Ruggieri et al.,1989), and it was shown subsequently that overexpressionof human RbAp48 and RbAp46 can also suppress the iralphenotype (Qian et al., 1993, 1995). To test whether tomatoLeMSII can suppress the phenotype of this mutant, wecloned the LeMSII cDNA into the yeast multicopy expressionvector pFL61. As shown in Figure 5, iral cells containingpFL61 alone did not survive heat shock, but cells transformedwith either yeast MSI1 or tomato LeMSII in pFL61 remainedviable, demonstrating that either gene can suppress the iralphenotype. We also transformed the same three plasmidsinto a ras2Val19 yeast strain. The ras2Val19 mutant has a re-duced intrinsic GTPase activity (Birchmeier et al., 1985) andalso exhibits heat shock sensitivity. Again, this phenotypewas suppressed by plasmids containing MSI1 or LeMSII

B

Figure 4. Immunolocalization of the LeMSII Protein in TomatoTissues.

(A) and (B) Immunolocalization in tomato fruit. Fruit with a diameterof 1 cm was sectioned and probed with the affinity-purified mouseanti-LeMSH antiserum (B) or the anti-LeMSH-depleted antiserum(A) followed by alkaline phosphatase-conjugated goat anti-mousesecondary antibody. The epidermis (e) and pericarp (p) are labeled.Magnification X54.(C) and (D) Immunolocalization in tomato floral meristems. Floralmeristems were sectioned and probed with the affinity-purifiedmouse anti-LeMSH antiserum (D) or the anti-LeMSM-depleted anti-serum (C) followed by alkaline phosphatase-conjugated goat anti-mouse secondary antibody. The meristem (m) and vascular tissue(v) are labeled. Magnification x 107.5.

but not by vector alone, indicating that tomato LeMSII isfunctionally homologous to the yeast gene in this assay.

LeMSII Binds Human RB and Maize RRB1

RbAp48 and RbAp46 originally were isolated from HeLa celllysates as proteins that specifically bound to a column con-taining the C terminus of the human RB protein (p56RB) (Leeet al., 1991; Qian et al., 1993, 1995). The interactions betweenRB and RbAp48 and RbAp46 are detectable both in vitro andin vivo (Qian et al., 1993, 1995). Because of the extensive se-quence similarity between human RbAp48 and RbAp46 and

1600 The Plant Cell

iral Strain(IR-1)

No Plasmid pFL61 (Vector)

ras2Va!19 strain(TK161-R2V)

No Plasmid pFL61 (Vector)

30°C

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No Plasmid pFL61 (Vector No Plasmid pFL61 (Vector)

Yeast MSI1 LeMSIIYeast MSI1 LeMSII

Figure 5. Suppression of Yeast Ras Pathway Mutations by Tomato LeMSII.

Cells of iral strain IR-1 or ras2Val19 strain TK161-R2V were transformed with the pFL61 vector alone, pFL61-LeMSI1, or pFL61-MSI (yeast MS/7).For growth under permissive conditions, transformed and untransformed cells were streaked and grown on minimal plates at 30°C. For growthunder nonpermissive conditions, transformed and untransformed cells were streaked on minimal plates and then heat shocked at 56°C for 25min (TK161-R2V) or 30 min (IR-1) before incubation at 30°C.

tomato LeMSII, we tested the ability of LeMSM to bind humanRB in an in vitro assay. Glutathione S-transferase (GST)-RbAp48 and GST-LeMSI1 fusion proteins were produced inEscherichia coli and purified by binding to glutathione-Sepharose beads. Fusion proteins were then incubated within vitro-translated p56RB and extensively washed, and boundfractions were analyzed by electrophoresis, as shown in Fig-ure 6A. Three bands are seen because of incomplete trans-lation or partial proteolysis of the in vitro-translated RBprotein. Under these conditions, the human RB fragmentsbind to both the human and tomato GST-RbAp48 and GST-LeMSh fusion proteins but not to the GST protein alone, in-dicating that the sequences necessary for this interactionhave been evolutionary conserved between the plant LeMSIIand mammalian RbAp48 and RbAp46 proteins.

We recently cloned cDNAs from maize encoding RB-likeproteins with regions of significant homology to the simianvirus 40 large T antigen binding domains of the human RB,p107, and p130 proteins (Ach et al., 1997). One cDNA en-codes a 96-kD full-length RB-like protein that we designatedRRB1. Two other groups have also isolated partial cDNAsfor RRB1 (Grafi et al., 1996; Xie et al., 1996). We testedwhether tomato LeMSM could bind to maize RRB1 by usingthe same assay in which we observed binding to human RB(Figure 6B). Both human RbAp48 and tomato LeMSM boundto in vitro-translated RRB1, whereas GST alone did not bindunder these conditions, indicating that the interaction be-tween RB-like proteins and the LeMSII, RbAp48, andRbAp46 WD-40 proteins has been conserved betweenplants and animals.


LeMSM Binds to a 65-kD Protein during Tomato FruitDevelopment

We have shown that LeMSH can interact with human RBand maize RRB1. To examine further the presence and dis-tribution of LeMSH binding proteins in tomato, we performedprotein-protein gel blot analysis using nuclear extracts fromvarious tomato tissues and a 32P-labeled GST-LeMSI1 pro-tein probe. We chose to focus most of our studies on nu-clear extracts from developing tomato fruit, because celldivision and expansion occur in distinct developmentalstages in tomato fruit (reviewed in Gillaspy et al., 1993) andthe binding of LeMSM to an RB-like protein suggests a po-tential role in cellular growth regulation.

Nuclear extracts were prepared (Manzara et al., 1991),fractionated on an SDS-polyacrylamide gel, and blotted tonitrocellulose. The filters were probed with GST-LeMSI1 fu-sion protein that had been 32P-labeled using heart musclekinase (Kaelin et al., 1992). Figure 7 shows that the GST-LeMSI protein binds a protein of ^65 kD (p65) from nuclearextracts from mature, ripe fruit. A small number of other lessabundant binding proteins was also detected with proteinextracts from red fruit, most notably in the 50-kD range. Wedid not observe any major binding activities with nuclear ex-tracts from fruit at other stages, except for three weak bandsof ~60, 70, and 110 kD in protein extracts from young fruit(0.2 to 0.8 cm). Protein binding was not detected under simi-lar conditions with nuclear protein extracts from tomato coty-ledons and mature leaves (data not shown). No binding was

Tomato Fruit

fco COo co

B

Human RB

Maize RRB1

Figure 6. Binding of Human RB and Maize RRB1 to HumanRbAp48 and Tomato LeMSH.

(A) Binding to human RB. Bacterially expressed GST, GST-p48 (humanRbAp48), and GST-LeMSI1 were purified by binding to glutathione-Sepharose beads. The in vitro-translated 35S-labeled C-terminalfragment of human RB was added to the bead-bound fusion pro-teins and incubated for 30 min. Beads were washed extensively, andbound proteins were eluted by boiling in SDS buffer and analyzed byelectrophoresis, followed by autoradiography.(B) Binding to maize RRB1. In vitro-translated, full-length maizeRRB1 was added to bacterially expressed GST, GST-p48, andGST-LeMSI1 bound to glutathione-Sepharose beads. Proteins wereincubated, washed, and analyzed as given in (A).

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Figure 7. Protein-Protein Gel Blot Analysis of LeMSH Binding Pro-teins in Tomato Fruit.

Nuclear extracts from various stages of tomato fruit developmentprepared from cultivar VFNT fruit were fractionated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. Filters wereprobed with the 32P-labeled GST-LeMSI1 fusion protein, washed,and autoradiographed.

observed with protein extracts from any tissue when labeledGST alone was used as a probe (data not shown).

We also probed a blotted maize endosperm nuclear pro-tein extract with our LeMSH probe (Figure 7) because weinitially isolated the maize RRB1 clone from a maize en-dosperm library. Two binding proteins of M50 to 65 kD weredetected in this extract as well as several smaller proteins.We did not detect a protein in the 96-kD range as expectedfor RRB1. However, we have found that bacterially ex-pressed RRB1 does not bind to the LeMSH protein underthese assay conditions (data not shown). This is probablybecause this assay requires the ability of the labeled proteinprobe to bind to denatured proteins in the blot; therefore,not all protein-protein interactions are detectable by thismethod. Thus, the p65 MSI binding proteins in tomato fruitand maize endosperm are probably not related to RRB andmay therefore represent a different class of MSI bindingproteins.

Because the p65 LeMSM binding protein was detected inripening tomato fruit, it is possible that its accumulation isdirectly or indirectly regulated by ethylene, a hormone thatcontrols many aspects of fruit ripening (reviewed in Fray andGrierson, 1993). To examine this possibility, we performedprotein-protein gel blot analysis with nuclear extracts fromthe mature fruit of two tomato fruit-ripening mutants, ripen-ing inhibitor (rin) and Neverripe (Nr). These mutants are bothdefective in initiating most aspects of the fruit-ripening pro-cess and have similar phenotypes. rin fruit mature veryslowly and do not accumulate lycopene. No ethylene is pro-duced at the onset of ripening, and normal ripening cannotbe induced by exogenous ethylene (Lincoln and Fischer,

1602 The Plant Cell

1988; Knapp et al., 1989). Nr fruit produce normal levels of ethylene, but they appear deficient in ethylene-inducible ripening processes such as polygalacturonase production (DellaPenna et al., 1989; Knapp et al., 1989). In the protein- protein gel blot assays using labeled LeMSll, nuclear extracts from fully grown wild-type, rin, and Nr tomato fruit all showed similar levels of p65 LeMSI1 binding activity (data not shown), indicating that the expression and accumulation of p65 in fruit development are most likely not regulated by ethylene-dependent processes.

DISCUSSION

RbAp48 and RbAp46 were originally isolated as major proteins from HeLa cell extracts that bound to an RB affinity column (Huang et al., 1991). This interaction subsequently was shown to occur both in vitro and in vivo (Qian et al., 1993, 1995). We show here that both Arabidopsis and tomato contain RbAp48-like proteins and that the tomato LeMSI1 protein can bind to the human RB protein. Because the LeMSIl and RbAp48 proteins share 65% sequence identity, the conservation of this interaction with RB is perhaps not surprising. However, we and others have recently cloned cDNAs encoding RB-like proteins from maize. These proteins have -30% identity to the mammalian RB protein in the simian virus 40 T antigen binding domains (Grafi et al., 1996; Xie et al., 1996; Ach et al., 1997). We show here that the maize RRBl protein can bind to the LeMSll protein in vitro, indicating that the ability of a physical interaction between these types of proteins has been conserved between the plant and animal kingdoms. The conservation of this association over such large evolutionaty distances argues for the functional importance of this interaction.

An Emerging Role for MSI-Type WD-40 Proteins in Chromatin Function

Recently, mammalian RbAp48 was found to be associated with histone deacetylase (Taunton et al., 1996), raising specu- lation that it serves as an adapter that targets the deacetylase to specific regions of chromatin. Because hypoacetylated histones are associated with inactive transcriptional domains (Wolffe and Pruss, 1996), perhaps the interaction of RbAp48 and MSI with RB helps to mediate or supplement some of the transcriptional repression effects of RB in mammalian cells (Cavanaugh et al., 1995; Weinberg, 1995; White et al., 1996) by targeting specific regions of the chromatin for deacetylation. In this way, RB-like proteins may have a function in the chromatin-dependent regulation of RNA polymerase II transcriptional activity, as they do in the recently reported general regulation of RNA polymerase I and 111 activities (reviewed in White, 1997).

Human RbAp48, like the Msil protein, is also a compo-

nent of the chromatin assembly complex (CAC), which pro- motes nucleosome formation using acetylated histones H3 and H4 (Verreault et al., 1996). In addition, the Hat2 protein in yeast is a subunit of a cytoplasmic histone acetyltransferase (Parthun et al., 1996). Because these related proteins are involved in severa1 aspects of histone metabolism, it has been suggested that they serve as histone chaperones during chromatin formation and maturation (Roth and Allis, 1996). Because the assembly and deposition of nucleosomes are critical features of S phase, it is possible that the binding of RbAp48 and MSI proteins by RB and RRBl helps to sequester the histone chaperones during G,, thus keep- ing aspects of nucleosome metabolism on hold until RB is phosphotylated at the restriction point and the cell is ready to begin DNA synthesis.

LeMSI1 and AtMSI1, AtMSIP, and AtMS13 Are Members of a Family of Conserved WD-40 Proteins

In Arabidopsis, maize (H. Feiler and W. Gruissem, unpublished data), humans, and yeast, the RbAp48 and MSI WD-40 proteins are encoded by small families of related genes, whereas in tomato, only one gene has been detected. It is possible that the different MSI family members have distinct functions. In humans, RbAp48 is associated with CAC, but RbAp46 is not (Verreault et al., 1996). Also, only a subset of RbAp48 is associated with CAC, and in glycerol gradients, the majority of RbAp48 sediments with higher molecular weight complexes. Although the function(s) of plant MSI proteins re- mains to be determined, it is possible that different MSI family members are components of distinct histone-modifying complexes varying in their functions and specificities.

In maize, biochemical studies have found multiple types of histone acetylase and deacetylase activities (Georgieva et al., 1991; Lopez-Rodas et al., 1991; Lechner et al., 1996). Interestingly, the HC toxin of the maize fungal pathogen Cochliobolus carbonum has been found to be a potent inhibitor of histone deacetylase activity (Brosch et al., 1995). If plant MSI proteins are components of plant histone deacetylases, then modulation of MSI levels in plants may create new possibilities for engineering fungal pathogen resistance.

We are currently using ectopic expression and antisense RNA mutagenesis approaches to determine the roles of the different MSI family members in Arabidopsis. Transgenic Arabidopsis plants with reduced levels of the AtMSI1 protein exhibit a range of mutant phenotypes, including vegetative and reproductive abnormalities, suggesting that MSI proteins are critical for normal plant growth and development (P. Taranto and W. Gruissem, unpublished data).

The yeast Msil protein is able to suppress mutations in the yeast Ras pathway that result in constitutively elevated levels of Ras-GTP, and it has been shown here and elsewhere (Qian et al., 1993, 1995) that the human and plant Msil homologs can also function to suppress these mutations in yeast. How- ever, the mechanism by which the MSI and RbAp48 proteins


suppress these mutations is not known. Although Ras has been consetved between mammals and yeast, there are significant differences in the downstream pathways that are regulated by Ras in each system. Intriguingly, Ras is essen- tia1 in mammalian cells for progression through the G1-to-S phase, and RB has been shown recently to be an essential mediator between the Ras signaling pathway and cell cycle activation (Peeper et al., 1997). However, it has not been demonstrated that RbAp48 or RbAp46 is involved in Ras signaling in mammalian cells, and no Ras homologs have been found in plants to date. Thus, it is possible that one or more of the roles of RbAp48 and RbAp46 in mammals, the MSI proteins in plants, and Msi l and Hat2 proteins in yeast are significantly different. It is also possible that the suppression of the phenotype of the Ras pathway mutations is caused by an alteration in the transcription of one of the genes in the Ras-cAMP pathway. Such alteration could be mediated by aberrant histone modification caused by overexpression of the MSI or RbAp proteins.

MSI Proteins Are Members of an Expanding Group of RB Binding Proteins in Plants

The plant MSI genes are the third type of gene cloned from plants that encodes proteins related to mammalian RB binding proteins. First, three genes have been cloned from Ara- bidopsis encoding cyclins that have homology to mammalian D cyclins and that contain LXCXE RB binding motifs at their N termini (Soni et al., 1995). We have shown elsewhere that one of these cyclins can bind to the maize RRB1 protein (Ach et al., 1997). Second, the wheat dwarf virus encodes a protein necessary for vira1 DNA replication. This protein can bind to mammalian RB (Collin et al., 1996) and the p130 protein (Xie et al., 1995) as well as to maize RRBl (Grafi et al., 1996; Xie et al., 1996). The tomato golden mosaic virus ALI protein, which is necessary for tomato golden mosaic virus DNA replication, also binds to the maize RRBl protein, although ALI does not contain the LXCXE binding motif (Ach et al., 1997). These interactions are reminiscent of the binding of the RB protein family in mammalian cells by the onco- proteins of the DNA tumor viruses. Third, we show that the tomato LeMSI1 protein, like human RbAp48 and RbAp46 (Qian et al., 1993, 1995), binds to mammalian RB as well as to maize RRBl. The presence of these three different types of RB binding proteins in plants suggests that many of the features of the RB system of cell cycle regulation have been conserved between kingdoms.

METHODS

Plant Materials

Tomato DNA, RNA, and protein extracts were from greenhouse- grown Lycopersicon esculenfum VFNT Cherry lA1221, except for

those from wild-type, ripening inhibitor (rin), and Neverripe (Nr) fruit, which were from cultivar Ailsa Craig. Tomato young fruit and Arabi- dopsis thaliana leaf cDNA libraries were as described by Ach and Gruissem (1994).

Degenerate Polymerase Chain Reaction and Cloning of Plant MSI cDNAs

For polymerase chain reaction (PCR) isolation of tomato LeMSll and Arabidopsis AtMSll fragments, two degenerate oligonucleotide primers were synthesized corresponding to amino acids 24 to 33 and 132 to 137 of the human RbAp48 protein (Qian et al., 1993). Their sequences are 5'-TGGAARAARAAYACTCCTTTYYTNTAYGA-3' (MF primer) and 5'-GGRTTYTGNGGCATRTA-3' (MR primer). PCR reactions with young tomato fruit and Arabidopsis leaf cDNA libraries were set up and performed as described by Ach and Gruissem (1994). For PCR isolation of an AtMSl3 fragment, the primer 5'- GGYTTYTGNGGCATRCA-3' used in the PCR along with MF primer, utilizing the Arabidopsis library as a substrate, were used.

PCR fragments were subcloned (TA cloning kit; Invitrogen, Carls- bad, CA) and sequenced. lnserts were 32P-labeled by random prim- ing (Prime-lt I1 kit; Stratagene, La Jolla, CA) and used to screen the cDNA libraries (Ach and Gruissem, 1994).

RNA and DNA Gel Blot Analyses

Total RNA was isolated from tomato tissues (Barkan et al., 1986), and 5-bg samples were blotted and hybridized (Narita and Gruissem, 1989) using a random primed 32P-labeled 160-bp Xbal probe from the LeMSll coding region. After autoradiography, filters were stripped and reprobed with a pea 18s nuclear rRNA gene probe (Ach and Gruissem, 1994). Gel blot analysis using tomato genomic DNA was performed (Ach and Gruissem, 1994) with the same probe that was used for the RNA gel blot analysis.

lmmunochemistry

Mouse anti-LeMSI1 polyclonal antibodies were produced by injecting mice with a thrombin-cleaved glutathione S-transferase (GST)-LeMSI fusion protein in Ribi adjuvant (Ribi Immunochemicals, Hamilton, MT), according to the manufacturer's protocol. The serum was subtrac- tively purified against a GST-expressing Escherichia coli extract and then affinity purified against a GST-LeMSI column, as described by Gillaspy et al. (1995). lmmunolocalizations were also performed according to Gillaspy et al. (1 995), except that tissues were fixed in 50% ethanol, 0.87 N acetic acid, and 3.7% formalin, and blocking and antibody reactions were performed in 1 % BSA in PBS. The mouse anti- LeMSl antibody (1 :1 O0 [v/v]) and alkaline phosphatase-conjugated goat anti-mouse antibodies (1:300 [v/v]; Boehringer Mannheim) were incubated for 12 hr at 4°C and 1 hr at 23"C, respectively. Levamisole (0.024% [w/v]; Sigma) was included in the alkaline phosphatase reaction to inhibit endogenous phosphatase activity, and color development proceeded for 4 hr. Anti-LeMSII-depleted antiserum was produced by incubating the anti-LeMSI1 antiserum with the GST- LeMSll fusion protein bound to glutathione-Sepharose 4B beads (Pharmacia Biotech, Piscataway, NJ). All sections were probed with the secondaty antibody alone as well as with the anti-LeMSI1- depleted antiserum to prove the specificity of the reactions.

1604 The Plant Cell

Yeast Transformation

The EcoRV-BamHI fragment of the LeMSl7 cDNA in the pBluescript SK- vector (Stratagene) and an EcoRV-Sphl fragment from the yeast MSl7 gene plasmid pd3 (Ruggieri et al., 1991) were cloned into yeast vector pFL61 (Minet et al., 1992) digested with Mscl. Saccharomyces cerevisiae strains IR-1 and TK161-R2V (Ruggieri et al., 1989) were transformed, plated onto SD plates, and grown at 30"C, as described in Ausubel et al. (1993). URA3 transformants were restreaked and grown at 30°C with or without a heat shock at 56°C for 25 min (TK161-R2VJ or 30 min (IR-1).

publication, Lisa Girard for the maize endosperm nuclear extract, Ann Loraine for mutant and wild-type Ailsa Craig fruit, Tim Durfee for comments on the manuscript, and members of the Gruissem laboratory for advice and comments. This work was supported by National Sci- ence Foundation Grant No. MCB 95-06985 to W.G. and a National Science Foundation Predoctoral Fellowship to P.T. R.A.A. was supported by a postdoctoral fellowship from Pioneer Hi-Bred lnternational and by National lnstitutes of Health Research Service Award No. GM14231.

Received April 15, 1997; accepted July 17, 1997

In Vitro Binding Assay with GST Fusion Proteins

The LeMSl7 coding region was isolated by PCR with the cDNA clone, using the primers 5'-CGCGGATCCGGGAAAGACGAAGAT- GAGATG-3' and 5'-CGGAATTCGTAGAGGGGCAAATAGAACAAG- 3'. After digestion with EcoRl and BamHI, the PCR product was cloned into EcoRI- and BamHI-digested pGEX-2TK (Kaelin et al., 1992). GST-RbAp48 (human) plasmid was a gift from E. Lee (Univer- sity of Texas, San Antonio).

GST fusion proteins were produced in E. coli and purified using glutathione-Sepharose, as described previously (Kaelin et al., 1991), except that cells were grown for 3 hr at room temperature after in- duction with isopropyl p-D-thiogalactopyranoside. Yield was quanti- tated on SDS-polyacrylamide gels. The human pRB56 fragment (Huang et al., 1991) and an equivalent region of maize RRBl were prepared by in vitro transcription, using T7 RNA polymerase (Ausubel et al., 1993). In vitro translation was then conducted with 35S- methionine (Amersham), using a rabbit reticulocyte kit (Promega).

For binding assays, purified GST fusion proteins bound to glutathione-Sepharose were washed three times in PBS and once in buffer A (50 mM Hepes, pH 7.7, 300 mM NaCl, and 0.1% Nonidet P-40). lncubation with in vitro-translated RB56 or RRB1 was performed for 30 min at room temperature in buffer A plus 10 mg/mL BSA. After incubation, the beads were washed five times with buffer A, boiled in SDS loading buffer, and fractionated on a 10% SDS- polyacrylamide gel, followed by autoradiography.

Protein-Protein Gel Blots

The GST-LeMSI1 fusion protein was labeled with heart muscle kinase (Sigma; Kaelin et al., 1992). Nuclear extracts were prepared from various tomato tissues (Mamara et al., 1991), and 8 Fg of each extract was fractionated on a 10% SDS-polyacrylamide gel and blotted onto nitrocellulose in 192 mM glycine, 25 mM Tris base, and 0.01 % SDS. Filters were prehybridized overnight at 4°C in 1 x HBB (20 mM Hepes, pH 7.5, 5 mM MgCI,, and 1 mM KCI), 0.05% Nonidet P-40, 1 mM DTT, and 5% nonfat dried milk. Filters were hybridized overnight at 4"C, using 100,000 to 250,000 cpm/mL labeled GST- LeMSll (or labeled GST alone as a negative control) in 1 x BB (20 mM Hepes, pH 7.5, 75 mM KCI, 0.1 mM EDTA, and 2.5 mM MgCI,) and 1% nonfat dried milk. Filters were washed for 30 min at room temperature in three changes of 1 x BB before autoradiography.

ACKNOWLEDGMENTS

We thank Rosamaria Ruggieri for the IR-1 and TKl61-R2V strains, Eva Lee for the GST-p48 plasmid and for communication of results before

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DOI 10.1105/tpc.9.9.1595 1997;9;1595-1606Plant Cell

R A Ach, P Taranto and W Gruissemanimals.

A conserved family of WD-40 proteins binds to the retinoblastoma protein in both plants and

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