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Identification and Characterization of Components of a Putative Petunia S-Locus F-Box–Containing E3 Ligase Complex Involved in S-RNase–Based Self-Incompatibility^[W]

^a Intercollege Graduate Degree Program in Plant Biology, Pennsylvania State University, University Park, Pennsylvania 16802
^b Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802

¹ To whom correspondence should be addressed. E-mail txk3{at}psu.edu; fax 814-863-7024.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF₂. We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S₁-, S₂-, and S₃-RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele–specific manner. Native glycosylated S₃-RNase is not degraded to any significant extent; however, deglycosylated S₃-RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF–containing E3 complex is unknown.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Self-incompatibility (SI) is an intraspecific reproductive barrier that allows pistils of a plant to reject self pollen (genetically related pollen) but to accept nonself pollen (genetically unrelated pollen) for fertilization (de Nettancourt, 2001). SI is further classified into two major types, sporophytic SI (SSI) and gametophytic SI (GSI), based on how pollen behavior in SI interactions is determined. In the simplest cases, both SSI and GSI are controlled by a highly polymorphic locus, named the S-locus, which contains two separate genes: one encoding female specificity and the other encoding male specificity (Takayama and Isogai, 2005). For SSI, the pollen phenotype is determined by the S-genotype of the pollen parent, and for GSI, the pollen phenotype is determined by the S-genotype of the pollen itself. In GSI, matching of the pollen S-allele with either S-allele of the pistil results in the inhibition of pollen tube growth in the style. Only pollen carrying a different S-allele from both S-alleles of the pistil can grow down through the style to effect fertilization in the ovary.

One type of GSI mechanism that has been studied extensively is the S-RNase–based SI, which has been found in the Solanaceae, Scrophulariaceae, and Rosaceae (Kao and Tsukamoto, 2004). In these families, the S-RNase gene, a highly polymorphic gene first identified in Nicotiana alata (Solanaceae) (Anderson et al., 1986), controls the pistil specificity in SI (Lee et al., 1994; Murfett et al., 1994). The RNase activity of S-RNases is required for their function in rejecting self pollen (Huang et al., 1994), and results consistent with rRNA degradation being responsible for growth inhibition of self pollen tubes have been obtained (McClure et al., 1990). S-RNases are glycoproteins with various numbers of N-linked glycan chains; however, the carbohydrate moiety is not required for their function in SI (Karunanandaa et al., 1994). Thus, the recognition function of S-RNases appears to reside in the protein backbone.

The S-Locus F-box (SLF) gene, which controls pollen specificity, was identified recently. Lai et al. (2002) first reported the identification of Ah SLF (Antirrhinum hispanicum SLF) from sequencing a 63-kb region of the S₂-locus of A. hispanicum (Scrophulariaceae) that contains the S₂-RNase gene. Subsequently, SLF (also named SFB for S-Locus F-Box) was identified in several species of the genus Prunus (Rosaceae). For example, Pm SLF (P. mume SLF) was identified from sequencing a 62.5-kb S₇-locus region (Entani et al., 2003), and Pd SFB (P. dulcis SFB) was identified from sequencing a 70-kb S_c-locus region of P. dulcis (Ushijima et al., 2003). In Petunia inflata (Solanaceae), Pi SLF was identified from sequence analysis of a 328-kb S-locus region that contains the S₂-RNase gene (Wang et al., 2004). The role of Pi SLF in SI was demonstrated by introducing its S₂-allele, Pi SLF₂, into S₁S₁, S₁S₂, and S₂S₃ plants and showing that the Pi SLF₂ transgene caused the breakdown of SI in pollen carrying the S₁- or S₃-allele but not in pollen carrying the S₂-allele (Sijacic et al., 2004). The rationale for this approach was based on the well-documented phenomenon called competitive interaction, in which pollen carrying two different S-alleles (heteroallelic pollen), but not pollen carrying two copies of the same S-allele (homoallelic pollen), fails to function in SI. Interestingly, introducing the S₂-allele of Ah SLF into Petunia hybrida S₃S₃ plants also caused the breakdown of SI in S₃ pollen that inherited the transgene (Qiao et al., 2004a), even though Ah SLF₂ is only 30% identical in amino acid sequence to Ph SLF₃ and Pi SLF₂. Several pollen-part mutants have been found to be associated with either truncation or deletion of SLF/SFB, strongly suggesting that this gene controls the pollen specificity in the Rosaceae (Ushijima et al., 2004; Sonneveld et al., 2005).

The finding that the pollen specificity gene encodes an F-box protein provides a clue to the biochemical mechanism of S-specific inhibition of pollen tube growth. Most F-box proteins are involved in ubiquitin-mediated protein degradation. This system uses E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin ligase) enzymes to catalyze the transfer of polyubiquitin chains to specific protein substrates for degradation by the 26S proteasome (Bai et al., 1996). One class of E3s is the RING-related class, which can be further divided into single subunit RING/U-box E3s and multisubunit E3s (Moon et al., 2004; Willems et al., 2004). SCF, a class of the multisubunit E3, consists of Skp1, Cullin-1, F-box protein, and Rbx1 (a RING-H2 protein) (Willems et al., 2004). Cullin-1 serves as the scaffold protein; its N- and C-terminal domains interact with Skp1 and Rbx1, respectively. The F-box domain of F-box proteins interacts with Skp1, and a separate domain interacts with specific protein substrates (Zheng et al., 2002). The substrate-interacting domains may contain WD40 repeats, Leu-rich repeats, other protein–protein interaction modules, or unrecognizable motifs (Cenciarelli et al., 1999; Moon et al., 2004). None of the SLFs characterized to date contains any known protein–protein interaction motifs in the C-terminal region (i.e., outside of the N-terminal F-box domain). Qiao et al. (2004b) have shown, by yeast two-hybrid, in vivo coimmunoprecipitation, and pull-down assays, that this C-terminal region of Ah SLF₂ interacts with both its self and nonself S-RNases with no allelic specificity. Huang et al. (2006) recently used yeast two-hybrid screens to identify a Skp1-like protein that interacted with the F-box domain of Ah SLF₂, suggesting that Ah SLF is likely to be a component of an SCF complex.

Using immunolocalization of pollinated pistils, Luu et al. (2000) showed that, in Solanum chacoense (Solanaceae), both self and nonself S-RNases were localized in the cytoplasm of the pollen tube. By contrast, Goldraij et al. (2006) reported that, in N. alata, both self and nonself S-RNases were sequestered in a vacuolar compartment of the pollen tube at an early stage (16 h after pollination) of pollen tube growth in the style. At a later stage (36 h after pollination), the compartment in the incompatible pollen tube was disrupted, presumably releasing S-RNases into the cytoplasm of the pollen tube to exert their cytotoxic function, whereas the compartment in the compatible pollen tube remained intact. However, Goldraij et al. (2006) could not rule out the possibility that some small amounts of self and nonself S-RNases taken up by the pollen tube were present in the cytoplasm. Thus, even if S-RNases are indeed compartmentalized after they are taken up by the pollen tube, it would appear that some mechanism should still be required to prevent nonself S-RNases from exerting their cytotoxic effect on pollen tubes.

A current model for the function of SLF is predicated on the assumption that SLF is a component of a canonical SCF complex (Qiao et al., 2004a, 2004b; Sijacic et al., 2004; Huang et al., 2006). This model predicts that an SLF interacts with its self and nonself S-RNases differently so that only nonself S-RNases are ubiquitinated and degraded by the 26S proteasome. As a first step toward testing the validity of this model in P. inflata, we set out to identify potential components of the putative SCF^{Pi SLF} complex. The results suggest that a Cullin-1 (named Pi CUL1-G), a RING finger protein (named Pi SBP1), and Pi SLF are likely to be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1 in the canonical SCF complex. The in vitro binding assay was also used to examine the interactions between S-RNases and Pi SLFs, and the results suggest that S-RNases interact with their nonself Pi SLFs to a greater extent than with their self Pi SLF and that Pi SLFs exhibit a similar binding property for S-RNases. Finally, we used pollen tube extracts and purified recombinant S₁-, S₂-, and S₃-RNases, as well as both glycosylated and deglycosylated forms of S₃-RNase purified from pistils, to show that all nonglycosylated S-RNases were degraded via the 26S proteasome pathway and that the recombinant S-RNases were ubiquitinated.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Isolation and Characterization of Three Skp1 Genes of P. inflata
To isolate Skp1 genes of P. inflata, we used cDNAs for ASK1 and ASK2, two well-characterized Arabidopsis thaliana Skp1s, as probes to screen an S₁S₁ pollen cDNA library under low-stringency hybridization conditions. Screening of 3 x 10⁵ plaque-forming units (pfu) resulted in four independent clones, and sequencing revealed that they all corresponded to the same gene. The longest cDNA was 681 bp, with a 468-bp open reading frame. The deduced amino acid sequence was 80 and 83% identical to the amino acid sequences of ASK1 and ASK2, respectively, suggesting that this cDNA encodes a Skp1. The corresponding gene was thus named Pi SK1 (for P. inflata SKP1). Genomic gel blot analysis revealed that, under low-stringency hybridization conditions, Pi SK1 hybridized to at least three additional genomic fragments of P. inflata (data not shown). To isolate other Skp1 genes homologous with Pi SK1, we used the full-length Pi SK1 cDNA as a probe to screen 3 x 10⁶ pfu of an S₂S₂ pollen cDNA library under low-stringency hybridization conditions. Twenty-two positive clones were isolated, and sequencing revealed that 6 encoded Pi SK1 and the other 16 corresponded to two Pi SK1 homologues. These two genes were named Pi SK2 and Pi SK3. Alignment of the amino acid sequences of Pi SK1, Pi SK2, and Pi SK3 with those of ASK1, ASK2, and a human SKP1 (Schulman et al., 2000) is shown in Supplemental Figure 1 online. Pair-wise amino acid sequence identities between these three P. inflata Skp1 proteins range from 90 to 92%, which is greater than the 79% amino acid sequence identity between ASK1 and ASK2.

We next used the yeast two-hybrid assay to examine whether Pi SK1, Pi SK2, and Pi SK3 interact with Pi SLF. The coding sequences of Pi SK1, Pi SK2, and Pi SK3 were inserted into a yeast two-hybrid bait vector, pGBD-C1 (James et al., 1996), and the coding sequence of Pi SLF₂, the product of the S₂-allele of Pi SLF, was inserted into a prey vector, pGAD-C1 (James et al., 1996). No positive interactions were observed between any of these three Pi SKs and Pi SLF₂ (Figure 1 ). The yeast two-hybrid assay was also performed using Pi SK1 and Pi SK2 in pGAD-C1 and Pi SLF₁ (for S₁-allele of Pi SLF) and Pi SLF₂ in pGBD-C1. Again, no interactions were observed in any of the four possible combinations of Pi SKs and Pi SLFs (data not shown). To ascertain whether these three Pi SKs are bona fide Skp1s, we used pGBD-C1-Pi SK1 as bait to screen an S₂S₂ pollen prey library previously constructed in pGAD424 (Skirpan et al., 2001). Twenty independent colonies were isolated under high-stringency screening. PCR fingerprinting and sequencing revealed that these 20 clones represented seven different genes, and the deduced amino acid sequences of all of them contained an F-box domain at the N terminus. ß-Galactosidase activity assays showed that all seven of these F-box proteins interacted strongly with Pi SK1, Pi SK2, and Pi SK3; the results for two of these F-box proteins, named Pi FBP23 and Pi FBP2011 (for P. inflata F-Box Protein 23 and 2011, respectively), are shown in Figure 1. The observation that all of the interacting proteins of Pi SK1 isolated from the yeast two-hybrid screen are F-box proteins suggests that Pi SK1 and its homologues, Pi SK2 and Pi SK3, are bona fide Skp1 proteins. None of the genes encoding these seven F-box proteins are likely linked to the S-locus, because no restriction fragment length polymorphism was observed when cDNAs for these genes were used as probes in genomic gel blot analysis of S₁S₁, S₂S₂, and S₃S₃ genotypes (data not shown).

View larger version (66K): [in this window] [in a new window]   Figure 1. Yeast Two-Hybrid Assay of Interactions between Three Pi SKs and Three F-Box Proteins. Pi FBP2011 and Pi FBP23 are F-box proteins that are likely encoded by genes unlinked to the S-locus. The bait vector is pGBD-C1, and the prey vector is pGAD-C1; p53 is a tumor suppressor and was used here as a negative control. Six colonies of yeast SFY526 coexpressing a pair of bait and prey proteins were streaked on filter paper for ß-galactosidase activity assay. The paper was incubated in an X-Gal–containing solution (Breeden and Nasmyth, 1985) for 1 h at 30°C.

Identification of Pi SBP1 as an Interacting Partner of Pi SLF
To further examine the possibility that Pi SLF might interact with other Skp1(s) present in the pollen tube, we used Pi SLF₂, Pi SLF₂(FB), and Pi SLF₂(CTD), which contains the C-terminal domain (amino acids 50 to 389), as baits to separately screen the S₂S₂ pollen prey library; 10, 13, and 7 positive colonies were isolated for pGBD-C1-Pi SLF₂, pGBD-C1-Pi SLF₂(FB), and pGBD-C1-Pi SLF₂(CTD), respectively. Restriction digestion and sequence analysis revealed that the cDNAs contained in all of the positive clones corresponded to the same gene whose deduced amino acid sequence was 98% identical with that of Ph SBP1 (for P. hybrida S-RNase Binding Protein1). Ph SBP1 had previously been identified by Sims and Ordanic (2001) from yeast two-hybrid screens using as baits truncated P. hybrida S-RNases (containing the two hypervariable regions, HVa and HVb, and the conserved region C3 but missing the conserved regions C1, C2, C4, and C5; defined in Ioerger et al., 1991). A Ph SBP1 homologue was identified in S. chacoense using the same approach (O'Brien et al., 2004). Because the amino acid sequence of the protein found to interact with Pi SLF₂ is almost completely identical to that of Ph SBP1, the protein was named Pi SBP1 (for P. inflata SBP1). An alignment of the amino acid sequences of Pi SBP1, Ph SBP1, and Sc SBP1 (for S. chacoense SBP1) is shown in Supplemental Figure 2 online. Amino acid sequence analysis by SMART showed that all three of these proteins have two protein–protein interaction domains: a coiled-coil region between amino acids 183 and 227, and a RING-HC domain between amino acids 289 and 323.

To determine whether Pi SLF₁ also interacts with Pi SBP1, pGAD-C1-Pi SBP1 was separately cotransformed with pGBD-C1-Pi SLF₁ and pGBD-C1-Pi SLF₂ into yeast. The interactions of Pi SBP1 with Pi SLF₁ and Pi SLF₂ were quantified by an o-nitrophenyl-ß-D-galactoside assay of the ß-galactosidase activity produced by the colonies (Figure 2A ). Comparable activities were detected for the interactions of Pi SBP1 with Pi SLF₁ and Pi SLF₂, whereas only background activities were detected in the negative controls.

View larger version (22K): [in this window] [in a new window]   Figure 2. Analyses of Interactions of Pi SBP1 with Pi SLF1 and Pi SLF2. (A) Yeast two-hybrid assay. Pi SBP1 (in prey vector pGAD-C1) along with three bait constructs containing Pi SLF1, Pi SLF2, or p53, and with the bait vector pGBD-C1, were separately cotransformed into the yeast strain SFY526, and the transformants were used for the assay. Pi SBP1 (in pGAD-C1) alone was also transformed into SFY526 (NONE). The ß-galactosidase activities shown are mean values + SD measured from six independent yeast transformants. (B) In vitro binding assay. Pi SLF2 and Pi FBP2411 (an F-box protein likely encoded by a gene unlinked to the S-locus) were expressed as (His)6:T7:Pi SLF2 and (His)6:T7:Pi FBP2411, respectively, and the purified proteins were incubated separately with GST:Pi SBP1-bound Glutathione Sepharose 4 Fast Flow resin. As negative controls in this figure and in Figures 4B, 6B, and 6C, the same amount of GST:Pi SBP1-bound resin used in each binding assay was incubated without any (His)6:T7-tagged protein, and GST-bound resin was incubated with the (His)6:T7-tagged protein, as indicated, under the same conditions used in each binding assay. The bound proteins (arrow) were eluted and analyzed by immunoblotting (IB) using an anti-(His)6 antibody. Input lanes in this figure and in Figures 4B, 5C, 5D, 6B, and 6C contain a fraction of the (His)6:T7-tagged protein, as indicated, used in the binding assay.

View larger version (15K): [in this window] [in a new window]   Figure 4. Analyses of Interactions of Pi SBP1 with Full-Size and Truncated S1- and S2-RNases. (A) Yeast two-hybrid assay. The assay was performed as described in the legend to Figure 2A. S1(HVabC3) and S2(HVabC3) are truncated S1- and S2-RNase, respectively, each containing the two hypervariable regions and the conserved C3 region. All of the negative controls are as described in the legend to Figure 2A. (B) In vitro binding assay. The interactions between GST:Pi SBP1 and purified (His)6:T7-tagged S1-RNase, S2-RNase, and RNase X2 were analyzed as described in the legend to Figure 2B. The bound proteins (arrow) were detected by immunoblotting (IB) using an anti-T7 antibody. The band above the (His)6:T7-tagged protein in each input lane is an E. coli protein that copurified with the tagged protein.

View larger version (19K): [in this window] [in a new window]   Figure 6. Interactions of Pi SBP1 with Pi CUL1-G and Ph UBC1. (A) Yeast two-hybrid assay showing that Pi SBP1, but not Pi RBX1, interacts with Pi CUL1-G, Ph UBC1 (an E2 ubiquitin-conjugating enzyme of P. hybrida), and Pi SLF2. The ß-galactosidase activity assay was performed as described in the legend to Figure 1 except that the filter paper was incubated for 5 h at 30°C. (B) In vitro binding assay of the interaction between Pi SBP1 and Pi CUL1-G. Purified (His)6:T7:Pi CUL1-G was tested for its interaction with GST:Pi SBP1 in the binding assay as described in the legend to Figure 2B. Top panel, immunoblot (IB). The bound (His)6:T7:Pi CUL1-G was detected by the anti-T7 tag antibody and is indicated with an arrow. The other bands with lower molecular masses cross-reacted with the antibody and were detected only after a long exposure of the blot. Bottom panel, Coomassie blue staining of a duplicate gel of that used in immunoblotting. The asterisk indicates the GST:Pi SBP1 band, and the double asterisks indicate the GST band. (C) In vitro binding assay of the interaction between Pi SBP1 and Ph UBC1. Purified (His)6:T7:Ph UBC1 was used along with GST:Pi SBP1 in the assay as described in the legend to Figure 2B. The bound (His)6:T7:Ph UBC1 was detected by an anti-T7 tag antibody. Top panel, immunoblot. The arrow indicates the (His)6:T7:Ph UBC1 band. Bottom panel, Coomassie blue staining of a duplicate gel of that used in immunoblotting. The asterisk indicates the GST:Pi SBP1 band, and the double asterisks indicate the GST band.

View larger version (47K): [in this window] [in a new window]   Figure 5. In Vitro Binding Assay of Interactions of Pi SLFs with S1-, S2-, and S3-RNases. (A) Interactions of Pi SLF1 and Pi SLF2 with S2-RNase. Equal amounts of purified (His)6:T7:Pi SLF1 and (His)6:T7:Pi SLF2 were assayed for their interactions with GST:S2-RNase, as described in the legend to Figure 2B, except that two independent reactions were performed to assess each interaction. Reaction mixtures containing GST-bound resin and the (His)6:T7-tagged protein, as indicated, were used as negative controls. Each input lane contains 10% of the amount of the (His)6:T7-tagged protein used in the binding reaction; note that the intensities of the (His)6:T7:Pi SLF1 and (His)6:T7:Pi SLF2 bands are approximately equal. Top panel, immunoblot (IB). The arrow indicates the (His)6:T7:Pi SLF1 band and the (His)6:T7:Pi SLF2 band detected by the anti-(His)6 tag antibody. Bottom panel, Ponceau S staining of a part of the blot shown in the top panel before immunoblotting to reveal equal amounts of GST:S2-RNase used in the binding assay. The single asterisk indicates the GST:S2-RNase band, and the double asterisks indicate the GST band. (B) Competition between Pi SLF1 and Pi SLF2 for interactions with S2-RNase. Equal amounts of purified (His)6:T7:Pi SLF1 and (His)6:T7:Pi SLF2 were mixed and used with GST:S2-RNase in the binding assay. Purified (His)6:T7:Pi SLF1 and (His)6:T7:Pi SLF2 were loaded in the control lanes to mark their respective positions on the gel. The reaction mixture containing GST-bound resin and both (His)6:T7-tagged proteins served as a negative control. The input lane contains 10% of the amount of the mixture of the two (His)6:T7-tagged proteins used in the binding reaction; note that the intensities of the (His)6:T7:Pi SLF1 and (His)6:T7:Pi SLF2 bands are approximately equal. (His)6:T7:Pi SLF1 (top band) and (His)6:T7:Pi SLF2 (bottom band) were detected by the anti-(His)6 tag antibody. (C) Interactions of Pi SLF2 with truncated S1- and S2-RNases. Purified (His)6:T7:Pi SLF2 was tested for its interaction with GST:S1(HVabC3) and GST:S2(HVabC3). The binding assay and negative control assays were performed as described in the legend to Figure 2B, except that GST:S1(HVabC3)-bound and GST:S2(HVabC3)-bound resins were used. The two truncated S-RNases are described in the legend to Figure 4A. Top panel, immunoblot. The arrow indicates the (His)6:T7:Pi SLF2 band detected by the anti-(His)6 tag antibody; the other bands with lower molecular masses cross-reacted with the antibody and were detected only after a long exposure of the blot. Bottom panel, Ponceau S staining of the blot shown in the top panel before immunoblotting; the single asterisk indicates the GST:S1(HVabC3) and GST:S2(HVabC3) bands, and the double asterisks indicate the GST band. (D) Interactions of Pi SLF1 with truncated S1- and S2-RNases. Except for the use of (His)6:T7-tagged Pi SLF1 and Pi FBP2411, the binding assay and negative control assays were performed as described for (C). The top and bottom panels are as described for (C), except that the arrow indicates the (His)6:T7:Pi SLF1 and (His)6:T7:Pi FBP2411 bands. (E) Binding differences between Pi SLF1 and S1- and S2-RNases. Different amounts of purified (His)6:T7:Pi SLF1 were used with equal amounts of GST:S1-RNase and GST:S2-RNase in separate binding assays, as described in the legend to Figure 2B. The top two panels show immunoblots against the anti-(His)6 tag antibody, and the bottom two panels show Ponceau S staining of the same blots before immunoblotting. (F) Binding differences between Pi SLF1 and S1- and S3-RNases. The assays were performed as described for (E) except that a different batch of purified (His)6:T7:Pi SLF1 and a different GST-tagged nonself S-RNase, GST:S3-RNase, were used.

Genomic Complexity and Expression Pattern of Pi SBP1
To determine whether Pi SBP1 exhibits S-specific restriction fragment length polymorphism, we used the full-length Pi SBP1 cDNA as a probe for genomic gel blot analysis of five different S-genotypes, S₁S₁, S₁S₂, S₂S₂, S₂S₃, and S₃S₃, of P. inflata (Figure 3A ). The same hybridizing fragment was detected in XbaI digests of all of the genotypes (no XbaI recognition sequence is present in the Pi SBP1 cDNA), and the same two hybridizing fragments were detected in EcoRI digests of all of the genotypes (one EcoRI recognition sequence is present in the Pi SBP1 cDNA). Thus, it is likely that Pi SBP1 is a single-copy gene and unlinked to the S-locus. RNA gel blot analysis showed that Pi SBP1 was expressed in all of the tissues examined (Figure 3B). All of these results are similar to those reported for Ph SBP1 by Sims and Ordanic (2001). Interestingly, the relative expression level of Pi SBP1 in anthers of different developmental stages and in pollen/pollen tubes is very similar to that of Pi SLF (Sijacic et al., 2004). The expression level peaked in stage 3 and stage 4 anthers, after the completion of meiosis of pollen mother cells to produce microspores (Lee et al., 1996), and was reduced significantly in mature pollen and in vitro–cultured pollen tubes (Figure 3B).

View larger version (60K): [in this window] [in a new window]   Figure 3. Genomic Hybridization and Expression Pattern of Pi SBP1. (A) DNA gel blot analysis. Genomic DNA (15 µg) isolated from each S-genotype indicated was digested with EcoRI or XbaI, and the blot was hybridized with the full-length Pi SBP1 cDNA. (B) RNA gel blot analysis. Each lane contained 20 µg of total RNA isolated from the tissue indicated, and the blot was hybridized with the full-length Pi SBP1 cDNA. The anther stages are defined by flower-bud size as described previously (Lee et al., 1996). Ethidium bromide staining of the gel used in blotting shows equal loading of the RNA samples.

The failure to detect any interaction between Pi SBP1 and S₁- and S₂-RNases by the yeast two-hybrid assay could be because S-RNase did not fold properly in the yeast cell, as it has eight conserved Cys residues involved in four intramolecular disulfide bonds (Ishimizu et al., 1996; Oxley and Bacic, 1996). Therefore, we used the in vitro binding assay, as described for Figure 2B, to reexamine the interaction. The coding sequences of S₁- and S₂-RNases were cloned into pET28 to produce (His)₆:T7:S₁-RNase and (His)₆:T7:S₂-RNase, respectively. As a control, the coding sequence (without the signal peptide; amino acid residues 1 to 193) of RNase X2 of P. inflata (Lee et al., 1992) was also cloned into pET28 to produce (His)₆:T7:RNase X2. RNase X2, an S-like RNase, is 43% identical to S₁- and S₂-RNases, and it was used to ascertain whether any interaction observed with either S-RNase is specific to S-RNase. The results showed that Pi SBP1 interacted with both S₁- and S₂-RNases but not with RNase X2 (Figure 4B).

S-RNases Interact with Nonself Pi SLFs to a Greater Extent Than with Self Pi SLFs, and Pi SLFs Show a Similar Binding Property for S-RNases
The in vitro binding assay described above was used to examine whether a Pi SLF interacts with its self S-RNase and/or nonself S-RNases, and if it interacts with both, whether there is any difference in the extent of the interaction. The coding sequences of S₁-, S₂-, and S₃-RNases were fused in-frame to the GST coding sequence in expression vector pGEX-5X-1 to produce GST:S₁-RNase, GST:S₂-RNase, and GST:S₃-RNase, respectively. The coding sequence of RNase X2 (without the signal peptide) was similarly fused to the GST coding sequence to produce GST:RNase X2. The coding sequence of Pi SLF₁ was cloned into pET28 to produce (His)₆:T7:Pi SLF₁. We first showed that both Pi SLF₁ and Pi SLF₂ interacted with S₁- and S₂-RNases but not with RNase X2 (see Supplemental Figure 3 online), suggesting that Pi SLFs specifically interact with S-RNases. To examine whether an S-RNase interacts with its self and nonself Pi SLFs to different extents, equal amounts of (His)₆:T7:Pi SLF₁ and (His)₆:T7:Pi SLF₂ were separately incubated with the same amount of resin-bound GST:S₂-RNase, and the bound proteins were detected by an anti-(His)₆ antibody. As shown in Figure 5A , the intensity of the (His)₆:T7:Pi SLF₁ band was much stronger than that of the (His)₆:T7:Pi SLF₂ band. The identity of these two bands was further confirmed by the anti-T7 antibody (data not shown). No binding was detected between GST and either (His)₆:T7:Pi SLF₁ or (His)₆:T7:Pi SLF₂ (Figure 5A). These results suggest that S₂-RNase interacts with its nonself Pi SLF to a greater extent than with its self Pi SLF. To further confirm this finding, we performed the in vitro binding assay in a single reaction mixture containing GST:S₂-RNase and equal amounts of (His)₆:T7:Pi SLF₁ and (His)₆:T7:Pi SLF₂ (Figure 5B). As these two (His)₆:T7-tagged proteins can be clearly separated by SDS-PAGE (see control lanes), we were able to assess the relative intensity of these two protein bands. Consistent with the results shown in Figure 5A, the intensity of the (His)₆:T7:Pi SLF₁ band was much stronger than that of the (His)₆:T7:Pi SLF₂ band.

We next examined whether Pi SLFs have a similar binding property for S-RNases, using the truncated S₁- and S₂-RNases that had been shown to interact with Pi SBP1 (Figure 4A). Purified GST:S₁(HV_abC3) and GST:S₂(HV_abC3) fusion proteins were used along with (His)₆:T7:Pi SLF₂ in the in vitro binding assay. As shown in Figure 5C, (His)₆:T7:Pi SLF₂ interacted with both truncated S-RNases, but the intensity of the (His)₆:T7:Pi SLF₂ band was stronger in the binding reaction containing GST:S₁(HV_abC3) than in that containing GST:S₂(HV_abC3). Moreover, (His)₆:T7:Pi SLF₁ interacted with both truncated S-RNases, but the intensity of the (His)₆:T7:Pi SLF₁ band was stronger in the binding reaction containing GST:S₂(HV_abC3) than in that containing GST:S₁(HV_abC3) (Figure 5D). These results suggest that both (His)₆:T7:Pi SLF₁ and (His)₆:T7:Pi SLF₂ interact with their respective truncated nonself S-RNases to a greater extent than with their respective truncated self S-RNases.

To determine whether the interaction between S-RNase and Pi SLF is specific to Pi SLF, we examined whether (His)₆:T7:Pi FBP2411 interacts with GST:S₁(HV_abC3) and GST:S₂(HV_abC3) in the in vitro binding assay. Pi FBP2411 was chosen because we had shown that both Pi FBP2411 and Pi SLF₂ interacted with Pi SBP1 (Figure 2B). No interactions were observed between (His)₆:T7:Pi FBP2411 and either of these two truncated S-RNases (Figure 5D), suggesting that S-RNases most likely interact specifically with Pi SLFs.

We performed additional in vitro binding assays to further assess the relative extent of binding between Pi SLF₁ and S₁-, S₂-, and S₃-RNases. In one assay, equal amounts of GST:S₁-RNase and GST:S₂-RNase were separately incubated with four different amounts of (His)₆:T7:Pi SLF₁, with each incubation in duplicate. Increasing binding of (His)₆:T7:Pi SLF₁ to both GST:S₁-RNase and GST:S₂-RNase was observed as the amount of (His)₆:T7:Pi SLF₁ was increased (Figure 5E). For all of the amounts examined, (His)₆:T7:Pi SLF₁ interacted with its nonself S-RNase, GST:S₂-RNase, to a greater extent than with its self S-RNase, GST:S₁-RNase. Most importantly, at the lowest amount used (2 µL), there was very little binding with GST:S₁-RNase but significant binding with GST:S₂-RNase. Another assay was performed similarly except that a different preparation of (His)₆:T7:Pi SLF₁ was used and that GST:S₃-RNase, instead of GST:S₂-RNase, was used as the nonself S-RNase. The results (Figure 5F) were similar to those shown in Figure 5E. For all of the amounts examined, (His)₆:T7:Pi SLF₁ interacted with its nonself S-RNase, GST:S₃-RNase, to a greater extent than with its self S-RNase, GST:S₁-RNase, and again at the lowest amount used (2 µL), there was very little binding of (His)₆:T7:Pi SLF₁ with GST:S₁-RNase but significant binding with GST:S₃-RNase.

Pi SBP1 Interacts with S-RNase and Pi SLF Differently
Because Pi SBP1 contains a coiled-coil region (amino acid residues 183 to 227; see Supplemental Figure 2 online) that could potentially be involved in protein–protein interactions, we examined whether this region is required for Pi SBP1 to interact with Pi SLF and S-RNase. The sequence for the coiled-coil region was deleted from the full-length Pi SBP1 cDNA to produce Pi SBP1(coiled-coil), which was cloned into pGAD-C1. pGAD-C1-Pi SBP1(coiled-coil) was separately cotransformed with pGBD-C1-S₁(HV_abC3), pGBD-C1-S₂(HV_abC3), and pGBD-C1-Pi SLF₂ into yeast for the two-hybrid assay. The results showed that Pi SBP1(coiled-coil) interacted with S₁(HV_abC3) and S₂(HV_abC3) but not with Pi SLF₂ (see Supplemental Figure 4 online). Thus, the coiled-coil region of Pi SBP1 is either directly or indirectly required for its interaction with Pi SLF₂ but is not required for its interaction with S-RNase, suggesting that S-RNase and Pi SLF interact differently with Pi SBP1.

Pi SBP1 Interacts with a Cullin-1 and an E2 Ubiquitin-Conjugating Enzyme
We isolated cDNAs for Cullin-1 and Rbx1 of P. inflata to examine whether they are components of the complex containing Pi SLF. A partial cullin-1 cDNA clone, previously isolated during our attempts to identify S-linked pollen-expressed genes by RNA differential display (J.A. Verica and T.-h. Kao, unpublished results; McCubbin et al., 2000), was used as a probe to screen 3 x 10⁵ pfu of the S₁S₁ pollen cDNA library and 6 x 10⁵ pfu of the S₂S₂ pollen cDNA library. Full-length cDNA clones for this gene, named Pi CUL1-C, were isolated from the S₂S₂ library, and full-length cDNA clones for another cullin-1 gene, named Pi CUL1-G, were isolated from both cDNA libraries. Pi CUL1-C and Pi CUL1-G shared 80% amino acid sequence identity, and among the five Arabidopsis Cullins (Moon et al., 2004), they are most similar to ATCUL1 (83 and 78% identity, respectively). The deduced amino acid sequences of Pi CUL1-C, Pi CUL1-G, ATCUL1, and a human CUL1 are aligned in Supplemental Figure 5 online. Because Pi CUL1-C and Pi CUL1-G are quite similar in sequence, we chose Pi CUL1-G for all subsequent studies. Screening of 6 x 10⁵ pfu of the S₂S₂ pollen cDNA library using the full-length cDNA for an Arabidopsis Rbx1, At RBX1 (Lechner et al., 2002), as a probe resulted in the isolation of one class of cDNA clones. The corresponding gene was named Pi RBX1; its deduced amino acid sequence is 87% identical to that of At RBX1. An alignment of the amino acid sequences of Pi RBX1, At RBX1, and a human Rbx1 is shown in Supplemental Figure 6 online.

The yeast two-hybrid assay showed that there were no interactions between Pi CUL1-G and Pi RBX1 (Figure 6A ) or between Pi RBX1 and Pi SLF₂ (Figure 6A). The findings that Pi RBX1 did not interact with either Pi CUL1-G or Pi SLF₂, and that Pi SBP1 interacted with Pi SLF₂ (Figures 2 and 6A), raised the possibility that Pi SBP1, but not Pi RBX1, is the RING protein that brings E2 into the complex containing Pi SLF. To address this possibility, we examined whether Pi SBP1 interacts with Pi CUL1-G and Ph UBC1, an E2 of P. hybrida. The yeast two-hybrid assay showed that Pi SBP1 interacted with both Pi CUL1-G and Ph UBC1 (Figure 6A). These interactions were further examined by the in vitro binding assay. The coding sequences of Pi CUL1-G and Ph UBC1 were cloned into pET28 to produce (His)₆:T7:Pi CUL1-G and (His)₆:T7:Ph UBC1, respectively. The results of the binding assay showed that GST:Pi SBP1 interacted with both (His)₆:T7:Pi CUL1-G (Figure 6B) and (His)₆:T7:Ph UBC1 (Figure 6C). The interactions of Pi SBP1 with a Cullin-1 and an E2 conjugating enzyme suggest that in the complex containing Pi SLF, Pi SBP1 plays the role of Rbx1 in the canonical SCF complex.

S-RNases Are Degraded via the 26S Proteasomal Pathway in a Non–S-Specific Manner in Pollen Tube Extracts
To examine whether pollen tubes contain a ubiquitin-26S proteasome pathway that degrades S-RNases, we developed a cell-free system using extracts of in vitro–germinated pollen tubes and purified GST:S-RNases or native S-RNases. First, GST:S₁-RNase and GST:S₂-RNase were incubated separately with extracts of in vitro–germinated S₂ pollen tubes at 30°C for 1 h in the absence or presence of MG132 (40 µM), a specific 26S proteasome inhibitor (Lee and Goldberg, 1998; Smalle and Vierstra, 2004). The proteins were then separated by SDS-PAGE, and an anti-GST antibody was used to detect GST:S₁-RNase and GST:S₂-RNase by protein gel blot analysis (Figure 7A ). Neither GST:S₁-RNase nor GST:S₂-RNase was detectable after 1 h of incubation in the absence of MG132. However, in the presence of MG132, the amount of each protein after 1 h of incubation was approximately the same as that before the incubation.

View larger version (52K): [in this window] [in a new window]   Figure 7. Degradation Assay of Bacterially Expressed GST, GST:S1-RNase, GST:S2-RNase, GST:S3-RNase, and GST:RNase X2, as Well as S3-RNase Purified from Pistils, by Extracts of S1, S2, or S3 Pollen Tubes. (A) GST:S1-RNase and GST:S2-RNase. Purified GST fusion proteins (0.3 µg each) were incubated separately with extracts of in vitro–germinated S2 pollen tubes in either the absence or presence of 40 µM MG132 (a specific inhibitor of the 26S proteasome) for 1 h at 30°C. An anti-GST antibody was used to detect GST:S1-RNase and GST:S2-RNase (arrow) as well as GST (double asterisks). IB, immunoblot. (B) GST:S3-RNase. Purified GST:S3-RNase (0.3 µg) was used in the assay as described for (A), except that both anti-GST and anti-S3-RNase antibodies were used to detect GST:S3-RNase. The arrow indicates GST:S3-RNase, the single asterisk indicates a cross-reacting protein present in the reaction mixture, and the double asterisks indicate GST. (C) Native glycosylated S3-RNase and its deglycosylated form. S3-RNase (0.1 µg) purified from pistils of the S3S3 genotype (left panel) and an equal amount of purified deglycosylated S3-RNase (right panel) were incubated separately with extracts of S1, S2, and S3 pollen tubes in either the absence or presence of MG132 (40 µM) for 90 min at 30°C. The anti-S3-RNase antibody was used to detect both glycosylated (open-headed arrow) and deglycosylated (closed arrows) S3-RNase. Single and double asterisks indicate cross-reacting proteins in the reaction mixture. A longer exposure time was used for the blot shown in the left panel (5 min) than that shown in the right panel (30 s). (D) GST:S3-RNase, GST:RNase X2, and GST. Each purified protein (0.3 µg) was used in the assay as described for (A). Left panel, immunoblot. The arrows indicate GST:S3-RNase or GST:RNase X2, and the double asterisks indicate GST. Right panel, Ponceau S staining of the blot shown in the left panel before immunoblotting.

Because native S-RNases are glycosylated but the bacterially expressed GST:S-RNases are not, we examined whether S₃-RNase purified from pistils of the S₃S₃ genotype was also degraded in pollen tube extracts. The purified native S₃-RNase was separately incubated with S₁, S₂, and S₃ pollen tube extracts, and the fate of the protein was analyzed by protein gel blotting using the anti-S₃-RNase antibody. The results (Figure 7C, left panel) showed that the native S₃-RNase was not degraded to a significant extent, if at all, after 90 min of incubation in the absence of MG132. However, when MG132 was present, a faint band (closed arrow) was detected in all three pollen tube extracts after the same length of incubation. Because this protein band was not detectable before the incubation and had a lower molecular mass than the native S₃-RNase band, we hypothesized that it might correspond to the deglycosylated S₃-RNase. That is, a small amount of S₃-RNase might have been deglycosylated and then degraded during the incubation in the pollen tube extracts without MG132.

To test this hypothesis, S₃-RNase purified from the pistils was treated with Peptide:N-Glycosidase F (PNGase F) to remove the N-linked glycan chain attached to Asn-29 (Karunanandaa et al., 1994) and then incubated with S₂ or S₃ pollen tube extract. The results of the degradation assay are shown in Figure 7C, right panel. The native S₃-RNase was completely deglycosylated by PNGase F (0-min lane), and the deglycosylated S₃-RNase band was virtually nondetectable after 90 min of incubation in either S₂ or S₃ pollen tube extract in the absence of MG132. However, the intensity of the deglycosylated S₃-RNase band after 90 min of incubation in the presence of MG132 was comparable to that at 0 min.

When a GST fusion protein is produced in Escherichia coli, the GST moiety of some fusion protein molecules is cleaved off around the Factor Xa cleavage site. This was also the case for GST:S₁-RNase, GST:S₂-RNase, and GST:S₃-RNase (Figures 7A and 7B). Under the assay conditions in which nonglycosylated S-RNases were completely degraded, GST molecules released from these recombinant S-RNases were not degraded. To further confirm that the degradation of nonglycosylated S-RNases in our in vitro system was not caused by general proteolytic cleavage, and to assess whether the degradation was specific to S-RNases, recombinant GST and GST:RNase X2 were examined, along with GST:S₃-RNase, in S₂ pollen tube extract. As shown in Figure 7D, GST was not degraded, whereas GST:RNase X2 was degraded in a similar 26S proteasome–dependent manner as GST:S₃-RNase. The findings that neither GST nor the native glycosylated S₃-RNase was degraded suggest that the degradation of nonglycosylated S-RNases was not caused by general proteases present in the pollen tube extracts. However, the degradation observed in our in vitro system was not S-specific, nor was it specific to S-RNases.

S-RNases Are Ubiquitinated by Pollen Tube Extracts in a Non–S-Specific Manner
Because the bacterially expressed nonglycosylated GST:S-RNases were degraded to similar extents in pollen tube extracts as the deglycosylated native S₃-RNase, we decided to use GST:S₂-RNase and GST:S₃-RNase to examine whether the degradation of S-RNase resulted from ubiquitination. Recombinant GST and GST:RNase X2 were used as controls to assess whether ubiquitination was restricted to the protein moiety (but not to the GST tag) and was specific to S-RNases. Using GST-tagged S-RNases and RNase X2 also allowed us to simplify the isolation of their ubiquitinated forms, if any, by affinity purification from among many other ubiquitinated proteins in pollen tube extracts (see Supplemental Figure 7 online). The ubiquitination assay was performed in a similar manner to the degradation assay except for the following modifications. First, 30 µg of ubiquitin or (His)₆:ubiquitin was added to S₂ pollen tube extract before incubation. Second, after incubation, GST:S₂-RNase, GST:S₃-RNase, GST:RNase X2, GST, and any products derived from them were isolated by Glutathione Sepharose 4 Fast Flow resin. The bound proteins were eluted and separated on two duplicated reducing SDS-polyacrylamide gels, and protein gel blotting was performed separately using the anti-GST antibody and an anti-ubiquitin or anti-(His)₆ antibody.

As shown in Figure 8A , after 5 min of incubation in the S₂ pollen tube extract at 30°C, the anti-GST antibody detected three discrete protein bands with molecular masses greater than that of GST:S₂-RNase, and all of the bands remained clearly visible after 10 min of incubation. One of these bands (Figures 8A and 8B, asterisks) was also detected by the anti-ubiquitin antibody at the 5- and 10-min time points (Figure 8B). Based on the estimated molecular masses of the three bands (67, 93, and 110 kD) detected by the anti-GST antibody, we attributed them to be GST:S₂-RNase (50 kD) conjugated with two, five, and seven ubiquitins (with total molecular masses of 67, 92.5, and 109.5 kD, respectively). After 1 h of incubation, the two bands with higher molecular masses had disappeared, but not the one possibly corresponding to GST:S₂-RNase conjugated to two ubiquitins. This ubiquitinated GST:S₂-RNase might have escaped degradation because the number of conjugated ubiquitin subunits was fewer than the minimum of four required for recognition by the 26S proteasome (Thrower et al., 2000). Moreover, the intensity of a protein band (Figure 8A, black dot) with a lower molecular mass than GST:S₂-RNase became more and more visible from the 5-min time point onward. This band could correspond to degradative product(s) of GST:S₂-RNase.

View larger version (59K): [in this window] [in a new window]   Figure 8. Ubiquitination Assay of GST, GST:S2-RNase, GST:S3-RNase, and GST:RNase X2 by Extracts of S2 Pollen Tubes. (A) and (B) Time course of ubiquitination and degradation of GST:S2-RNase. GST:S2-RNase (0.5 µg) was used in each reaction containing ubiquitin, and the reaction was stopped at different time points as indicated. The anti-GST antibody was used in the protein gel blot shown in (A), and an anti-ubiquitin antibody was used in the blot shown in (B). The open triangles indicate GST:S2-RNase, and the closed triangle indicates GST. The black dot indicates the degradative products of GST:S2-RNase. The closed arrows indicate three ubiquitinated forms of GST:S2-RNase with two, five, and seven ubiquitin subunits, and the asterisks indicate GST:S2-RNase conjugated with five ubiquitin subunits that was detected by both anti-GST and anti-ubiquitin antibodies. IB, immunoblot. (C) and (D) Ubiquitination assay of GST, GST:S3-RNase, and GST:RNase X2. Purified GST, GST:S3-RNase, and GST:RNase X2 (0.5 µg each) were used in the assay as described for (A) and (B), except that the reaction contained (His)6:ubiquitin and was analyzed only at the 10-min time point. Each reaction mixture was then divided equally and electrophoresed on two duplicate gels. The transferred membranes were immunoblotted with the anti-GST antibody (C) and the anti-(His)6 antibody (D). Top panels, immunoblot. Bottom panels, Ponceau S staining of the blots shown in the top panels before immunoblotting.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
The recent identification of the SLF gene as the pollen S-gene has opened up opportunities to investigate the biochemical mechanism of S-RNase–based SI. As the RNase activity of S-RNases is required for their function in SI (Huang et al., 1994) and rRNA of pollen tubes may be degraded after incompatible pollination (McClure et al., 1990), S-RNases are thought to act as cytotoxic molecules to inhibit the growth of self pollen tubes through RNA degradation. Both Luu et al. (2000) and Goldraij et al. (2006) have shown that uptake of S-RNases by pollen tubes is not S-allele–specific. Before the identification of SLF as the pollen S-gene, one of the models proposed that the pollen S-allele product encodes a cytosolic RNase inhibitor, which specifically inhibits the RNase activity of all nonself S-RNases and renders them nonfunctional inside a pollen tube (Kao and McCubbin, 1996; Golz et al., 2001). Because most F-box proteins are involved in ubiquitin-mediated protein degradation, this model has now been modified to state that a pollen S-allele product mediates the degradation of its nonself S-RNases, but not that of its self S-RNase, inside a pollen tube (Qiao et al., 2004b; Sijacic et al., 2004; Huang et al., 2006). According to this model, pollen S-allele products regulate the stability, rather than the RNase activity, of S-RNases. In this work, we have begun to address the biochemical role of the SLF of P. inflata.

The Complex That Contains Pi SLF Is Not a Canonical SCF Complex
If Pi SLF functions as a conventional F-box protein, it would be expected to be a component of an SCF complex. In this work, we have obtained several lines of evidence to suggest that the Pi SLF–containing complex is a not a canonical SCF complex and instead is a novel E3 ligase complex containing a Cullin-1 protein (Pi CUL1-G), a RING-HC protein (Pi SBP1), and Pi SLF but not containing Skp1 or Rbx1. First, we isolated cDNAs for three Skp1s of P. inflata and showed that none of them interact with Pi SLF₂ in the yeast two-hybrid assay (Figure 1) or the in vitro binding assay (data not shown). When Pi SK1, one of these Skp1s, was used as bait in yeast two-hybrid library screening, all seven classes of prey proteins identified were F-box proteins, suggesting that Pi SK1, and most likely its homologues, Pi SK2 and Pi SK3, are bona fide Skp1s. Second, the Arabidopsis genome is predicted to encode 19 Skp1s, but none of the 7 Skp1s representing all seven subgroups interact with Pi SLF₂ in the yeast two-hybrid assay. Third, Pi CUL1-G interacts with Pi SBP1 but not with Pi RBX1 (Figures 6A and 6B). Fourth, Pi SLF₁ and Pi SLF₂ interact with Pi SBP1 (Figure 2) but not with Pi RBX1 (Figure 6A). Fifth, Pi SBP1 interacts with an E2 conjugating enzyme (Figure 6C). Interestingly, the expression of both Pi SLF and Pi SBP1 peaks during pollen development and declines significantly in mature pollen and in vitro–germinated pollen tubes (Figure 3B) (Sijacic et al., 2004). It is possible that the protein produced in developing microspores is retained in mature pollen and pollen tubes.

We have concluded that, in the Pi SLF–containing complex, Pi SBP1 likely plays the roles of Skp1 and Rbx1 of a canonical SCF complex. This is because Pi SBP1, like Skp1, bridges the Cullin-1 component (Pi CUL1-G) and an F-box protein (Pi SLF) and, like Rbx1, interacts with the Cullin-1 component and E2. Pi SBP1 (335 amino acids) is approximately three times the size of Pi RBX1 (116 amino acids), so it could potentially interact with more proteins than does Pi RBX1. Variants of the SCF complex have been reported that contain some but not all of the typical components (Willems et al., 2004). For example, an SCF-like complex in humans consists of Skp1, an F-box protein (Ebi), a RING-HC protein (Siah-1), and an adaptor protein (SIP) that bridges Siah-1 and Skp1 (Matsuzawa and Reed, 2001). In this complex, Siah-1 plays the roles of Cullin-1 and Rbx1, and interestingly, Siah-1 (298 amino acids) is also larger than Rbx1. Another example is the E2F1 transcription factor, which can be ubiquitinated by multiple ROC-Cullin ligases that do not contain Skp1 (Ohta and Xiong, 2001).

Moreover, in Drosophila melanogaster, Sina, a RING-HC protein, forms an E3 ligase complex with Phyllopod (Phyl) and Ebi to regulate the degradation of a transcription repressor protein, Tramtrack (Ttk) (Li et al., 2002). In this complex, Ebi interacts with Sina and the substrate, Ttk. Sina and Phyl can mediate the degradation of Ttk, but Ebi promotes a more efficient degradation of Ttk, perhaps by stabilizing the Sina-Phyl-Ttk complex (Boulton et al., 2000; Li et al., 2002). Because SBP1 could potentially function as a single-subunit E3, the finding by Sims and Ordanic (2001) that Ph SBP1 interacted with S-RNases posed a conundrum concerning why two E3s (Ph SBP1 and the then predicted SLF-containing SCF complex) would be involved in the ubiquitination of S-RNases (McClure, 2004). Our finding that Pi SBP1 is a component of a novel E3 ligase complex containing Pi SLF and that Pi SBP1 likely assumes the roles of Skp1 and Rbx1 of a canonical SCF complex would provide a solution to this conundrum. Moreover, because Pi SBP1 alone interacts with S-RNases, the Pi CUL1-G-Pi SBP1-Pi SLF complex may function in a similar manner as the Sina-Phyl-Ebi complex in that Pi CUL1-G-Pi SBP1 could mediate the basal degradation of all S-RNases and Pi SLF could promote the efficient degradation of specific S-RNases (see below).

Our finding that Skp1 is not a component of the Pi SLF–containing complex contradicts the results for Ah SLF of A. hispanicum. Qiao et al. (2004b) used an anti-Ah SLF₂ antibody to pull down protein complexes that contained Ah SLF₂ from mixtures of pollen and stylar proteins under both compatible and incompatible conditions. They found proteins that cross-reacted with an anti-ASK1 antibody or with an anti-ATCUL1 antibody. Huang et al. (2006) subsequently used yeast two-hybrid screens to identify a Skp1-like protein, named Ah SSK1, that interacted with the F-box domain of Ah SLF₂. Thus, the Ah SLF–containing complex of A. hispanicum may have a different subunit composition from that of the Pi SLF–containing complex of P. inflata. In this context, it is interesting that the SLF of the Solanaceae functions differently at the mechanistic level from the SLF/SFB of the Rosaceae, as all of the pollen-part self-compatible mutants characterized to date in the Solanaceae resulted from duplication, not deletion, of the pollen S-allele (SLF) (Golz et al., 1999, 2001), whereas those in the Rosaceae resulted from defects (deletion or frame-shift mutations) in SLF/SFB (Ushijima et al., 2004; Sonneveld et al., 2005). Moreover, in the Solanaceae, pollen carrying two different functional S-alleles (i.e., SLF alleles) fails to function in SI as a result of competitive interaction (Golz et al., 2001; Sijacic et al., 2004), whereas in sour cherry (Prunus cerasus) of the Rosaceae, such heteroallelic pollen has been shown to function normally in SI (Hauck et al., 2006). Thus, it appears that even though all three of these families use F-box proteins and S-RNases as the male and female determinants, respectively, the biochemical mechanisms of SI are likely to have diverged.

S-RNases Are Degraded via a Ubiquitin-26S Proteasomal Pathway
If the Pi SLF–containing E3 ligase complex is involved in the degradation of S-RNases in pollen tubes, one would expect the degradation to be via the ubiquitin-mediated 26S proteasomal pathway. Previously, Qiao et al. (2004b) addressed the role of this protein degradation pathway in SI of Antirrhinum by examining the effect of 26S proteasome inhibitors on the in vitro growth of pollen tubes in the presence of either compatible or incompatible stylar extracts. They found that the inhibitors had no effect on the growth inhibition of pollen tubes by incompatible stylar extracts but inhibited the growth of pollen tubes in compatible stylar extracts by 50%. Qiao et al. (2004b) further showed that protease inhibitors had no significant effect on the growth of compatible pollen tubes; thus, they concluded that protein degradation by the 26S proteasome is required for the growth of compatible pollen tubes. The ubiquitin-mediated 26S proteasomal pathway is thought to be involved in many developmental processes. For example, >5% of the Arabidopsis proteome is related to this pathway (Smalle and Vierstra, 2004). Thus, inhibitors of the 26S proteasome will likely affect a plethora of biochemical events during pollen tube growth, complicating the interpretation of results.

Another approach to address the role of the 26S proteasome in SI is to compare the amounts of S-RNase in pistils after compatible pollination with those after incompatible pollination at various times after pollination, up to the time when the growth of incompatible pollen tubes is inhibited in the style. For example, Qiao et al. (2004b) pollinated S₂S₅ pistils of Antirrhirum with compatible or incompatible pollen, isolated total proteins from pistils collected at eight different time points (up to 60 h) after compatible or incompatible pollination, and determined the amounts of S₂-RNase. They detected a lower amount of S₂-RNase in compatibly pollinated pistils than in incompatibly pollinated pistils at 36 h after pollination, and they interpreted the results to mean that S₂-RNase was degraded in compatible pollen tubes. Goldraij et al. (2006) used a similar approach to measure the amounts of S_C10-RNase of N. alata in S_C10S_C10 pistils that had been pollinated with S_C10 or S₁₀₅ pollen, but they did not detect any significant difference in the amounts of S_C10-RNase between compatibly and incompatibly pollinated pistils up to 48 h after pollination. Because S-RNase is abundant in the style and it is very likely that not all of the S-RNase molecules are taken up by pollen tubes, it is difficult, if not impossible, to know precisely how much of the total amount determined from pollinated pistils is contributed by the S-RNase inside pollen tubes. This may be the reason why Qiao et al. (2004b) and Goldraij et al. (2006) reached opposite conclusions about whether S-RNase is degraded in compatible pollen tubes.

Goldraij et al. (2006) also dissected pollen tubes from S_A2S_A2 pistils pollinated with compatible or incompatible pollen and determined the amounts of S_A2-RNase in soluble and membrane fractions of both compatible and incompatible pollen tubes. They concluded that there was no significant difference in the amounts of S_A2-RNase between the soluble or membrane fractions of compatible and incompatible pollen tubes. Accurate determination of the amount of S-RNase inside pollen tubes by this approach requires the isolation of intact pollen tubes completely free of contaminating style tissue, which is abundant in S-RNase. This may be technically difficult, as the pollen tube preparations of Goldraij et al. (2006) also contained transmitting tract cells and extracellular matrix material of the style tissue. Another potential problem of using pollinated pistils or dissected pollen tubes to examine the fate of S-RNase is that if the great majority of S-RNase taken up by a pollen tube is indeed sequestered in a vacuolar compartment and not subject to degradation, as suggested by Goldraij et al. (2006), it would be difficult to accurately assess the degradation of a small amount of S-RNase among a large amount of sequestered S-RNase.

Here, we have developed a cell-free system to examine whether S-RNases are degraded in compatible pollen tubes, and if so, whether this is mediated by the ubiquitin/26S proteasomal pathway. The system involves the use of extracts of in vitro–germinated pollen tubes and exogenously added GST:S-RNases or native S-RNases purified from pistils. As the ubiquitin-mediated 26S proteasome degradation machinery is active in pollen tube extracts, we were able to use this cell-free system to show that the bacterially expressed GST:S₁-RNase, GST:S₂-RNase, GST:S₃-RNase, and GST-RNase X2, but not GST alone, were degraded by S₂ pollen tube extracts (Figures 7A, 7B, and 7D). Moreover, we found that the degradation was dependent of the activity of the 26S proteasome, as MG132, a specific inhibitor of the 26S proteasome, completely inhibited the degradation (Figures 7A, 7B, and 7D). We also showed that GST:S₂-RNase, GST:S₃-RNase, and GST-RNase X2, but not GST alone, were rapidly ubiquitinated in this cell-free system [with the addition of ubiquitin or (His)₆:ubiquitin] (Figure 8). The time course of ubiquitination of GST:S₂-RNase suggested that the molecules conjugated with four or more ubiquitins were subsequently degraded (Figure 8A and 8B). The use of the native glycosylated S₃-RNase in the degradation assay led us