PSI of the Colonial Alga Botryococcus braunii Has an Unusually Large Antenna Size

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

The multisubunit-pigment-protein complex PSI is an essential component of the electron transport chain in oxygenic photosynthetic organisms. It utilizes solar energy in the form of visible light to transfer electrons from plastocyanin to ferredoxin.PSI consists of a core complex composed of 12 to 14 proteins, which contains the reaction center (RC) and ∼100 chlorophylls (Chls), and a peripheral antenna system, which enlarges the absorption cross section of the core and differs in different organisms (Mazor et al., 2017; Iwai et al., 2018; Pi et al., 2018; Suga et al., 2019; for reviews, see Croce and van Amerongen, 2020; Suga and Shen, 2020). For the antenna system, cyanobacteria use water-soluble phycobilisomes; green algae, mosses, and plants use membrane-embedded light-harvesting complexes (LHCs); and red algae contain both phycobilisomes and LHCs (Busch and Hippler, 2011). In the core complex, PsaA and PsaB, the subunits that bind the RC Chls, are highly conserved, while the small subunits PsaK, PsaL, PsaM, PsaN, and PsaF have undergone substantial changes in their amino acid sequences during the evolution from cyanobacteria to vascular plants (Grotjohann and Fromme, 2013). The appearance of the core subunits PsaH and PsaG and the change of the PSI supramolecular organization from trimer/tetramer to monomer are associated with the evolution of LHCs in green algae and land plants (Busch and Hippler, 2011; Watanabe et al., 2014).A characteristic of the PSI complexes conserved through evolution is the presence of “red” forms, i.e. Chls that are lower in energy than the RC (Croce and van Amerongen, 2013). These forms extend the spectral range of PSI beyond that of PSII and contribute significantly to light harvesting in a dense canopy or algae mat, which is enriched in far-red light (Rivadossi et al., 1999). The red forms slow down the energy migration to the RC by introducing uphill transfer steps, but they have little effect on the PSI quantum efficiency, which remains ∼1 (Gobets et al., 2001; Jennings et al., 2003; Engelmann et al., 2006; Wientjes et al., 2011). In addition to their role in light-harvesting, the red forms were suggested to be important for photoprotection (Carbonera et al., 2005).Two types of LHCs can act as PSI antennae in green algae, mosses, and plants: (1) PSI-specific (e.g. LHCI; Croce et al., 2002; Mozzo et al., 2010), Lhcb9 in Physcomitrella patens (Iwai et al., 2018), and Tidi in Dunaliela salina (Varsano et al., 2006); and (2) promiscuous antennae (i.e. complexes that can serve both PSI and PSII; Kyle et al., 1983; Wientjes et al., 2013a; Drop et al., 2014; Pietrzykowska et al., 2014).PSI-specific antenna proteins vary in type and number between algae, mosses, and plants. For example, the genomes of several green algae contain a larger number of lhca genes than those of vascular plants (Neilson and Durnford, 2010). The PSI-LHCI complex of plants includes only four Lhcas (Lhca1–Lhc4), which are present in all conditions analyzed so far (Ballottari et al., 2007; Wientjes et al., 2009; Mazor et al., 2017), while in algae and mosses, 8 to 10 Lhcas bind to the PSI core (Drop et al., 2011; Iwai et al., 2018; Pinnola et al., 2018; Kubota-Kawai et al., 2019; Suga et al., 2019). Moreover, some PSI-specific antennae are either only expressed, or differently expressed, under certain environmental conditions (Moseley et al., 2002; Varsano et al., 2006; Swingley et al., 2010; Iwai and Yokono, 2017), contributing to the variability of the PSI antenna size in algae and mosses.The colonial green alga Botryococcus braunii (Trebouxiophyceae) is found worldwide throughout different climate zones and has been targeted for the production of hydrocarbons and sugars (Metzger and Largeau, 2005; Eroglu et al., 2011; Tasić et al., 2016). Here, we have purified and characterized PSI from an industrially relevant strain isolated from a mountain lake in Portugal (Gouveia et al., 2017). This B. braunii strain forms colonies, and since the light intensity inside the colony is low, it is expected that PSI in this strain has a large antenna size (van den Berg et al., 2019). We provide evidence that B. braunii PSI differs from that of closely related organisms through the particular organization of its antenna. The structural and functional characterization of B. braunii PSI highlights a large flexibility of PSI and its antennae throughout the green lineage.     

Structure versus function: Are new conformations of pannexin 1 yet to be resolved?

Pannexin 1 (Panx1) plays a decisive role in multiple physiological and pathological settings, including oxygen delivery to tissues, mucociliary clearance in airways, sepsis, neuropathic pain, and epilepsy. It is widely accepted that Panx1 exerts its role in the context of purinergic signaling by providing a transmembrane pathway for ATP. However, under certain conditions, Panx1 can also act as a highly selective membrane channel for chloride ions without ATP permeability. A recent flurry of publications has provided structural information about the Panx1 channel. However, while these structures are consistent with a chloride selective channel, none show a conformation with strong support for the ATP release function of Panx1. In this Viewpoint, we critically assess the existing evidence for the function and structure of the Panx1 channel and conclude that the structure corresponding to the ATP permeation pathway is yet to be determined. We also list a set of additional topics needing attention and propose ways to attain the large-pore, ATP-permeable conformation of the Panx1 channel.

IntroductionInitially, the pannexin field got off to an inauspicious start. Because the three proteins of the pannexin family (Panx1, Panx2, and Panx3) were discovered on the basis of their limited sequence homology to the invertebrate innexin gap junction proteins, it was assumed that they, too, would form gap junctions (Panchin et al., 2000; Bruzzone et al., 2003). However, notwithstanding some disputed science, it soon became clear that the gap junction function of pannexins is not realized (Dahl and Locovei, 2006; Huang et al., 2007; Sosinsky et al., 2011). Gap junction formation by pannexins is moreover prevented by glycosylation of the Panx1 protein (Boassa et al., 2007; Penuela et al., 2007; Boassa et al., 2008). It is conceivable, however, that the nonglycosylated form of Panx1 could allow the docking of oligomers in apposing membranes to each other. Indeed, such docking was observed in cryo-EM preparations of a mutant Panx1, N255A (Ruan et al., 2020). However, because glycosylation of the protein is required for membrane trafficking (Boassa et al., 2007; Penuela et al., 2007), it is unlikely that this process occurs in vivo. This notion is supported by the observation that a glycosylation mutant of Panx1 failed to lead to junctional conductance between paired oocytes, while expression of wtPanx1 followed by glycosidase F treatment before pairing of the cells resulted in gap junction formation, albeit at a much reduced rate as compared with the rate observed with connexins (Boassa et al., 2008). Furthermore, the gap junction function also is unlikely because of theoretical reasons and experimental findings, including expression in single cells, such as erythrocytes (Locovei et al., 2006a), and exclusive expression in the apical membrane of polarized epithelial cells (Ransford et al., 2009; Hanner et al., 2012; Shum et al., 2019). Of the three pannexins, only Panx1 has been studied in detail, and a clear channel function is well established for it. Therefore, this Viewpoint will exclusively deal with Panx1.It is generally assumed that an important function of Panx1 is to form ATP release channels. For example, as of this writing, a PubMed search with “pannexin” and “ATP release” as search terms yielded 287 publications. However, the biophysical evidence for this function is sparse, and the supporting evidence is largely correlative and/or based on pharmacological or genetic interference with ATP release. The ATP release function of Panx1 channels has been contested based on evidence that the channels are highly selective for Cl⁻ and the lack of detectable ATP release under the experimental conditions, i.e., activation by voltage, used in these studies (Ma et al., 2012; Romanov et al., 2012).It was long held that the oligomeric state of the Panx1 channel is that of a hexamer (Penuela et al., 2013; Dahl, 2015; Chiu et al., 2018). Recently, in a <6-mo period, six research groups independently published similar cryo-EM structures of the Panx1 membrane channel (Deng et al., 2020; Jin et al., 2020; Michalski et al., 2020; Mou et al., 2020; Qu et al., 2020; Ruan et al., 2020). All these papers challenge the view of the oligomeric state of the channel. Instead of the hexameric arrangement of identical Panx1 subunits, the channel appears to be formed by a homomeric heptamer.It is uncontested that the Panx1 channel can operate in a conformation in which the channel exhibits a unitary conductance of <100 pS. However, it has also been reported that, under certain experimental conditions, the channel can exhibit several subconductances with very rare sojourns to a maximal conductance of ∼500 pS (Dahl 2015).Activation of the Panx1 channel can be obtained by various stimuli, some physiological or pathological, others in the form of experimental tools. The majority of the physiological stimuli involve ligands, including ATP, glutamate, α adrenergic agonists, and bradykinin, binding to their cognitive receptors and leading to opening of Panx1 channels and “secondary” ATP release (Dahl 2015, 2018). A nonreversible activation of the channel is induced by cleavage with caspase 3 or 8 (Chekeni et al., 2010), leading to cell death. Experimental stimulation can be obtained in some cells but not in others by increasing the extracellular K⁺ concentration (Silverman et al., 2009; Wang and Dahl 2018; Chiu et al., 2018).Here, we present a point of view on these contested issues in the Panx1 field and attempt to reconcile apparently contradictory data or their interpretations. In addition, we ask to what extent the new structural data support the functional data.The structure of Panx1 channels as revealed by cryo-EMThe excitement for the long-awaited Panx1 structure was rewarded with not one but six publications, all appearing within the first half of 2020 and using cryo-EM to image the channel (Deng et al., 2020; Jin et al., 2020; Michalski et al., 2020; Mou et al., 2020; Qu et al., 2020; Ruan et al., 2020). Five of the six Panx1 publications showed human Panx1 structures (Deng et al., 2020; Jin et al., 2020; Michalski et al., 2020; Mou et al., 2020; Qu et al., 2020; Ruan et al., 2020), and two showed frog Panx1 structures (Deng et al., 2020; Michalski et al., 2020). To stabilize the frog Panx1 structure, thus improving the resolution for cryo-EM, Michalski et al. (2020) truncated the C terminus by 71 amino acids and removed 24 amino acids from the intracellular loop between transmembrane (TM) helices 2 and 3; this truncation was the version reported. The other publications reported full-length Panx1 structures, although one used a truncated Panx1 variant (Ruan et al., 2020) and another used a mutation (Jin et al., 2020) as their principal references. Because the C terminus–truncated structure and the (combined) WT structure had the highest overall quality of the several Panx1 structures presented by Ruan et al. (2020), it was used for de novo model building, as the reference structure for analyses, and for discussion. For the same reason, Jin et al. (2020) used the double mutation D376E/D379E, which eliminated the caspase-cleavage site, for their de novo model building. The overall architecture and dimensions of the human and frog Panx1 structures were similar, and the extracellular domains (ECDs) and transmembrane domains (TMDs) were nearly identical, except that the N-terminal helix (NTH) of the frog Panx1 is positioned on the intracellular side and not within the TMD as reported in human Panx1. These structures are becoming an important and critical tool to guide functional experiments and open a new venue to perform molecular dynamics simulations.The most novel finding shared by all the Panx1 structures is that instead of the hexamer hitherto believed to represent the native state of the Panx1 channel, the cryo-EM data show unequivocally a homo-heptamer arranged around a central symmetry axis that constitutes the principal permeation pathway. Panx1’s structural envelope adopts an inverted pail shape in the view parallel to the membrane. The heptameric channel is ∼110 Å long and ∼100 Å wide, with a flat ECD protruding ∼35 Å above the cell membrane and an intracellular domain (ICD) extending ∼35 Å into the cytoplasm with the TMD in between (Fig. 1). Both the N and C termini reside on the cytoplasmic side of the channel. Each TMD has four membrane-spanning helices per protomer. The TMD pore is predominantly lined by hydrophobic amino acids. The NTH is short and lines the TMD pore (human Panx1) with the NTH of one subunit interacting with the TMD of the adjacent subunit. Thus, it has been proposed that the NTH helps to maintain a rigid TMD pore (Ruan et al., 2020). Both the extracellular and intracellular pore entrances are lined with positively charged amino acids, making them favorable for negatively charged cargos such as Cl⁻ and ATP to enter and leave the pore.Open in a separate windowFigure 1.Structural model of Panx1. (A) Cartoon representation of Panx1 WT (PDB accession no. 6wbk) stabilized with detergent. The approximate position of the membrane is indicated by the bars. One protomer of the heptameric assembly is indicated in orange. The lipids resolved in the structure are colored in magenta. It is notable that many of the resolved lipids are found at the interface between subunits. CTH, C-terminal helix; CLH, cytoplasmic linker helix. (B) Overlay of different Panx1 cryo-EM–based structures from different groups (PDB accession no. 6lto = gold, WT hPanx1; PDB accession no. 6wbk = blue, hPanx1 ΔC terminus, ΔN terminus; PDB accession no. 6v6d = lilac, WT hPanx1). The structures diverge very little in the ECD and the membrane domain. However, the variation is larger in the ICD. The approximate positions of F54 and I58 are indicated. (C) Comparison of the extracellular pore. Key residues are annotated using their one-letter abbreviation. All structures are shown as backbone traces except W74, which is part of the constriction ring of the pore. The narrowest constriction is indicated by the arrow.A cap structure is formed on the extracellular side from seven ECDs, one from each subunit. The ECDs are organized into the pore’s most constricted site, thus defining the maximum size of permeable molecules and establishing the extracellular entrance to the TMD. Each ECD contains one helix cross-linked through a disulfide bond to a three-stranded antiparallel β-sheet. The N-terminal end of each subunit helix protrudes inward toward the central pore axis contributing to the constriction site. The key residues involved in the gating of Panx1 are part of the W74-R75-D81 inter-subunit triplet (Fig. 1) from the N-terminal ends of the ECD helices and are proposed to provide rigidity to the extracellular entrance containing positive charges (Deng et al., 2020). This ring lining the wall of the constriction defines a hydrated pore diameter of ∼9 Å (Fig. 1) and is the crux of the argument that the extracellular constriction site is the effective selectivity filter discriminating cargoes on the basis of their charge and size. By contrast, the narrowest point of the pore in the TMD has a hydrated diameter of ∼13 Å.Other inter-subunit interactions that stabilize the pore arrangement in the TMD are (1) the F67-Y79 aromatic–aromatic and Q266-T252 hydrogen bonding in the ECD, (2) the TM helix TM1-TM1 and TM2-TM4 interfaces between adjacent subunits, (3) the N-terminal loop from the neighboring subunit extending into the TMD pore lining, and (4) the resolvable residues of C-terminal helix 3 association with the cytoplasmic linker helices 1 and 2 from the adjacent subunit (Fig. 1; Deng et al., 2020; Michalski et al., 2020). In addition, membrane lipids occupy the crevice between TM3 and an adjacent TM4 at the edge of the channel. Lipid head groups associate with positively charged residues from the N-terminal end of TM3 (K214) and the C-terminal domain of TM4 (R302 and K303; Deng et al., 2020). It may be that the lipid environment influences the assembly and possibly the activation of the Panx1 channel.The pore substantially widens toward the intracellular side. The ICDs extend away from the pore axis, producing a voluminous intracellular vestibule. Two helices in the ICD connect to two TMD helices to form a compact assembly that provides rigidity to the vestibule. The vestibule is slightly larger in the frog Panx1 structure, but the pore architecture is essentially the same as in the human version. The C-terminal segment consisting of ∼70 amino acids, including the caspase cleavage site located immediately before the C terminus, was not visualized in any of the published cryo-EM structures, suggesting that it is intrinsically flexible. C-terminal cleavage removes its autoinhibitory effect without inducing an overall conformational change to the pore (Jin et al., 2020).A novel feature presented by Ruan et al. (2020) is seven narrow side tunnels in the upper ICD running perpendicular to and ending at the pore, like a T-intersection. This tunnel network was proposed to allow passage of small anions because each tunnel contains several positively charged and other polar residues along its length. The flexible NTH-TM1 linker would function as a gate. They hypothesized that these side tunnels facilitate Cl⁻ flux when the pore is blocked by the C terminus residing in the ICD vestibule. Movement or cleavage of the C terminus then would induce the ATP-permeable large-pore conformation. Further structural work is needed to determine whether the C terminus actually resides in the ICD vestibule. One complication with this tunnel hypothesis is that each tunnel has two constriction sites formed by R29 and A33, with diameters of 5.8 and 4.0 Å, respectively, meaning that partial dehydration of a Cl⁻ ion is likely required for it to pass through the tunnel. The possibility of anion tunnels in the Panx1 structure raises the question: could there be multiple open (active) conformations, and does “open” mean open to ATP and Cl⁻ ion or Cl⁻ ion only? Three of the six papers (Michalski et al., 2020; Qu et al., 2020) assume that the Panx1 structure is in the closed or inactive conformation, whereas the other three report an open or active conformation. These discrepancies should be resolved with further work. Interestingly, the overlay of three of these structures, one presumably open and two closed, shows nearly identical conformations of the extracellular constriction of the pore (Fig. 1 C).The structures of Panx1 truncation and mutant variants, as well as its docked inhibitor, carbenoxolone (CBX), illuminate the roles of distinct structural elements. For example, cryo-EM revealed that CBX sits atop the ECD and plugs the W74-R75-D81 ring (Jin et al., 2020; Ruan et al., 2020), supporting pore blocking as its mechanism of Panx1 inhibition. Second, removal of the NTH did not much change the shape of the TMD. This truncation still generated a CBX-sensitive, voltage-dependent current similar to the full-length Panx1 current (Ruan et al., 2020), suggesting that the NTH may play a role in the assembly of the heptamer from the Panx1 protomers rather than being an essential element in the pore conduction pathway. Third, the Panx1 structures obtained in the presence of Ca²⁺ and K⁺ were indistinguishable from the untreated Panx1 structure, suggesting that neither Ca²⁺ nor K⁺ is likely to directly activate the Panx1 channel (Ruan et al., 2020). Fourth, the glycosylation-deficient mutant (N255A) generated gap junction structures as well as hemichannels as determined by cryo-EM (Ruan et al., 2020). The N255A gap junction is formed by two hemichannels docked by the interaction of the extracellular linker 2 on apposing ECDs. Ruan et al. (2020) noted that this Panx1 gap junction likely is not a normal physiological conformation.Comparing the Panx1 structure with other ATP-permeant pore structures offers a blueprint for dissecting pore properties of Panx1. The oligomeric configuration of the ATP-permeant channels does not appear to correlate with pore diameter, in particular when comparing the narrowest extent of the pore (Table 1). Like Panx1, CALHM1 was originally thought to adopt a hexameric configuration (Siebert et al., 2013) until the cryo-EM structure revealed an octameric configuration (Syrjanen et al., 2020). It should be noted that not all connexin channels transport ATP. For example, Cx26, Cx30, Cx43, and Cx46 transport ATP, but Cx32 does not (Harris, 2001; Hansen et al., 2014). An interesting comparison can be made between Panx1 and human Cx31.3. Cx31.3 has a pore with a constriction site diameter of ∼8 Å, similar to Panx1’s constriction of ∼9 Å, and also selectively transports Cl⁻ and ATP (Lee et al., 2020). One difference between the two structures is that the constriction is in the ICD’s NTH for Cx31.3 and in the ECD for Panx1. Lee et al. (2020) pointed out that because Cx31.3’s pore constriction at the cytoplasmic entrance is smaller than the effective hydrated diameter of ATP (∼12 Å; Sabirov and Okada, 2004), the reported conformation would not allow ATP to pass through the pore without substantial conformational changes of the NTH. The same argument could be applied to Panx1’s ECD.Table 1.Comparison of oligomeric configuration with pore constriction diameter

Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry ‘Lapins’

Recent studies have shown that loss of pollen-S function in S₄′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S₄-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S₄-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S₄-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S₄-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

In sweet cherry (Prunus avium), self-incompatibility is mainly controlled by the S-locus, which is located at the end of chromosome 6 (Akagi et al., 2016; Shirasawa et al., 2017). Although the vast majority of sweet cherry varieties show self-incompatibility, some self-compatible varieties have been identified, most of which resulted from the use of x-ray mutagenesis and continuous cross-breeding (Ushijima et al., 2004; Sonneveld et al., 2005). At present, naturally occurring self-compatible varieties are rare (Marchese et al., 2007; Wünsch et al., 2010; Ono et al., 2018). X-ray-induced mutations that have given rise to self-compatibility include a 4-bp deletion (TTAT) in the gene encoding an SFB4′ (S-locus F-box 4′) protein, located in the S-locus and regarded as the dominant pollen factor in self-incompatibility. This mutation is present in the first identified self-compatible sweet cherry variety, ‘Stellar’, as well as in a series of its self-compatible descendants, including ‘Lapins’, ‘Yanyang’, and ‘Sweet heart’ (Lapins, 1971; Ushijima et al., 2004). Deletion of SFB3 and a large fragment insertion in SFB5 have also been identified in other self-compatible sweet cherry varieties (Sonneveld et al., 2005; Marchese et al., 2007). Additionally, a mutation not linked to the S-locus (linked instead to the M-locus) could also cause self-compatibility in sweet cherry and closely related species such as apricot (Prunus armeniaca; Wünsch et al., 2010; Zuriaga et al., 2013; Muñoz-Sanz et al., 2017; Ono et al., 2018). Much of the self-compatibility in Prunus species seems to be closely linked to mutation of SFB in the S-locus (Zhu et al., 2004; Muñoz-Espinoza et al., 2017); however, the mechanism of how this mutation of SFB causes self-compatibility is unknown.The gene composition of the S-locus in sweet cherry differs from that of other gametophytic self-incompatible species, such as apple (Malus domestica), pear (Pyrus spp.), and petunia (Petunia spp.). In sweet cherry, in addition to a single S-RNase gene, the S-locus contains one SFB gene, which has a high level of allelic polymorphism, and three SLFL (S-locus F-box-like) genes with low levels of, or no, allelic polymorphism (Ushijima et al., 2004; Matsumoto et al., 2008). By contrast, the apple, pear, and petunia S-locus usually contains one S-RNase and 16 to 20 F-box genes (Kakui et al., 2011; Okada et al., 2011, 2013; Minamikawa et al., 2014; Williams et al., 2014a; Yuan et al., 2014; Kubo et al., 2015; Pratas et al., 2018). The F-box gene, named SFBB (S-locus F-box brother) in apple and pear and SLF (S-locus F-box) in petunia, exhibits higher sequence similarity with SLFL than with SFB from sweet cherry (Matsumoto et al., 2008; Tao and Iezzoni, 2010). The protein encoded by SLF in the petunia S-locus is thought to be part of an SCF (Skp, Cullin, F-box)-containing complex that recognizes nonself S-RNase and degrades it through the ubiquitin pathway (Kubo et al., 2010; Zhao et al., 2010; Chen et al., 2012; Entani et al., 2014; Li et al., 2014, 2016, 2017; Sun et al., 2018). In sweet cherry, a number of reports have described the expression and protein interactions of SFB, SLFL, Skp1, and Cullin (Ushijima et al., 2004; Matsumoto et al., 2012); however, only a few reports have examined the relationship between SFB/SLFL and S-RNase (Matsumoto and Tao, 2016, 2019), and none has investigated whether the SFB/SLFL proteins participate in the ubiquitin labeling of S-RNase.Although the function of SFB4 and SLFL in self-compatibility is unknown, the observation that S4′ pollen tubes grow in sweet cherry pistils that harbor the same S alleles led us to speculate that S4′ pollen might inhibit the toxicity of self S-RNase. In petunia, the results of several studies have suggested that pollen tubes inhibit self S-RNase when an SLF gene from another S-locus haplotype is expressed (Sijacic et al., 2004; Kubo et al., 2010; Williams et al., 2014b; Sun et al., 2018). For example, when SLF2 from the S₇ haplotype is heterologously expressed in pollen harboring the S₉ or S₁₁ haplotype, the S₉ or S₁₁ pollen acquire the capacity to inhibit self S-RNase and break down self-incompatibility (Kubo et al., 2010). The SLF2 protein in petunia has been proposed to ubiquitinate S₉-RNase and S₁₁-RNase and lead to its degradation through the 26S proteasome pathway (Entani et al., 2014). If SFB/SLFL in sweet cherry have a similar function, the S4′ pollen would not be expected to inhibit self S₄-RNase, prompting the suggestion that the functions of SFB/SLFL in sweet cherry and SLF in petunia vary (Tao and Iezzoni, 2010; Matsumoto et al., 2012).In this study, we used sweet cherry to investigate how S4′ pollen inhibits S-RNase and causes self-compatibility, focusing on the question of whether the SFB/SLFL protein can ubiquitinate S-RNase, resulting in its degradation.