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.2011 Apr 29;145(3):371-82.
doi: 10.1016/j.cell.2011.03.021.

A conserved F box regulatory complex controls proteasome activity in Drosophila

Affiliations

A conserved F box regulatory complex controls proteasome activity in Drosophila

Maya Bader et al. Cell..

Abstract

The ubiquitin-proteasome system catalyzes the degradation of intracellular proteins. Although ubiquitination of proteins determines their stabilities, there is growing evidence that proteasome function is also regulated. We report the functional characterization of a conserved proteasomal regulatory complex. We identified DmPI31 as a binding partner of the F box protein Nutcracker, a component of an SCF ubiquitin ligase (E3) required for caspase activation during sperm differentiation in Drosophila. DmPI31 binds Nutcracker via a conserved mechanism that is also used by mammalian FBXO7 and PI31. Nutcracker promotes DmPI31 stability, which is necessary for caspase activation, proteasome function, and sperm differentiation. DmPI31 can activate 26S proteasomes in vitro, and increasing DmPI31 levels suppresses defects caused by diminished proteasome activity in vivo. Furthermore, loss of DmPI31 function causes lethality, cell-cycle abnormalities, and defects in protein degradation, demonstrating that DmPI31 is physiologically required for normal proteasome activity.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1. Proteomic screen for Nutcracker interacting proteins
[A] Scheme for testis-specific interactors of Nutcracker; PrA-nutcracker (PrA-ntc) was specifically expressed in testes with the Don-Juan (DJ) promoter. Testes were dissected, lysed, and incubated with IgG beads. Interacting complexes were eluted off the beads, and identified by either mass-spectrometry or Western-blot analysis.[B] SDS-PAGE commassie blots of Nutcracker interactors from cell lysate. PrA-ntc or PrA-ntc&DeltaF were expressed in S2 cells and used to make lysates for co-immunoprecipitation (co-IP) assays. Non-PrA expressing cells were used as a negative control. An arrow marks the DmPI31 band. See also Figure S1.[C] PrA-ntc is associated with proteasomesin vivo. PrA-ntc was expressed in testes and used for co-IP assays. Probing with an antibody against the proteasome subunit alpha7 shows that PrA-ntc forms a complex with proteasome proteinsin vivo.[D] The Nutcracker-DmPI31 interaction is not dependent on the F-box domain. PrA-ntc or PrA-ntcΔF were expressed in testes and used to make lysates for co-IP assays. Non-PrA expressing wild-type testes (yw) were used as a negative control. DmPI31 binding was detected by Western-blot analysis using a DmPI31 antibody.[E] Nutcracker-DmPI31 binding is mediated by a specialized domain. A conserved valine in Nutcarcker that mediates the interaction between the human PI31 and the F-box protein FBXO7 was mutated to investigate its importance for DmPI31-Nutcracker binding. PrA-ntc or PrA-ntcV-E were expressed in S2 cells and lysates were used for co-IP. Non-PrA expressing cells were used as a negative control.
Figure 2
Figure 2. DmPI31 is localized to Individualization Complexes
[A] Multiple alignments of DmPI31 and three mammalian homologues (bovine, mouse, and human). DmPI31 shares overall 45% homology with the mammalian proteins, corresponding to 29% identity and 16% similarity. In particular, the DX7H and YXLXY motifs, which are important for the PI31 structure, are highly conserved (labeled in yellow and green, respectively). Highlighted in blue are amino acids that mediate PI31-FBXO7 binding (Kirk et al., 2008). In pink is the Hydrophobic-Tyrosine-X (HbYX) motif. All accession numbers are listed in the M&M.[B] DmPI31 mRNA is abundant in adult testes. Semi-quantitative RT-PCR of dmPI31 mRNA transcripts taken from whole-body adult females, wild-type males orson-of-oskar males, which lack germ-cells. These are also compared to mRNA transcripts from wild-type testes orson-of-oskar testes. Beta-tubulin primers were used as control for total mRNA concentrations. Shown is cycle 25, 5 cycles before saturation.[C] Schematic diagram of spermatid individualization. An actin-based individualization complex (IC, red) forms around the elongated nuclei of 64 spermatids (gray) that are connected by cytoplasmic bridges. As the IC moves, cytoplasm (green) and organelles are collected in the cystic bulge (CB) and eventually discarded in the waste bag (WB), generating individual sperm devoid of most cytoplasm and organelles.[D] Caspase staining of individualizing cysts. Wild-type cysts were stained with DAPI (nuclei, blue), phalloidin (IC, red) and anti-active-caspase-3 (cytoplasm, green).[E–G] DmPI31 and Nutcracker localize to the same sub-cellular region during individualization.[E] Nutcracker staining during individualization as detected with an antibody that specifically recognizes this protein (Bader et al.). Nutcracker staining was seen at the base of the nuclei when the actin cones form around it (inset), and in a circular pattern around the cones in the CB (asterisks).[F] DmPI31 antibody staining. Like Nutcracker, DmPI31 protein is detected at the base of the elongated nuclei (inset), and co-localizes in a circular pattern with the actin cones (asterisks). Colocalization was also seen in cultured cells (Figure S2)[G] mCherry-DmPI31 fusion protein localization during individualization. This fusion protein is expressed under the control ofdmPI31 endogenous promoter and localized in a pattern virtually identical to DmPI31 antibody staining. mCherry-DmPI31 is seen in the combined cytoplasm of each cyst and at the base of fully elongated nuclei (inset), co-localizing with the actin cones as they move down the cyst (asterisks).
Figure 3
Figure 3. Nutcracker controls caspase activation and sperm individualization by promoting DmPI31 stability
[A] Mutations innutcracker affect DmPI31 stability. Testes lysates of the indicated genotypes were used to detect steady-state DmPI31 protein levels. The cleaved form of DmPI31 found innutcracker mutants is indicated by an arrow. The lowest molecular weight band is unspecific and serves as loading control.yw (wild type), ms771+/− (nutcrackerms771 heterozygote), ms771−/− (nutcrackerms771 homozygote), ms771(−/−) Rescue, (nutcrackerms771;hsp83-nutcracker), ms07259 (nutcracker07259 homozygote).[B] Mutations innutcracker result in the C-terminal cleavage of DmPI31. Total lysates from either wild-type ornutcrackerms771−/− homozygote flies expressing mCherry-DmPI31 were used to detect the size of the fusion protein after cleavage. The diagram explains the expected molecular weights, which differ depending on whether the cleavage is N- or C-terminal. The observed cleavage fragment was ~50KD, and clearly not ~37KD. This indicates that the C-terminal domain of DmPI31 is truncated innutcracker mutants.[C] The cleaved form of DmPI31 can physically associate with Nutcracker. PrA-ntc was expressed in testes of eithernutcrackerms771+/− ornutcrackerms771−/− background and used to make lysates for co-IP. The cleaved form of DmPI31 found innutcracker homozygote mutants is indicated by an arrow. Non-PrA expressing (yw) testes were used as a negative control.[D] DmPI31 stability depends on Nutcracker binding. Testes lysates fromnutcrackerms771−/− homozygote mutants expressing the indicated rescue constructs were used to detect steady-state DmPI31 protein levels. hsp83-nutcracker and hsp83-nutcrackerV-E rescue constructs are labeled wt and V-E respectively. The cleaved form of DmPI31 found innutcracker mutants is indicated by an arrow. This cleavage is not dependent on caspase or proteasome protease activity (Figure S3A). The lowest molecular weight band is unspecific and serves as loading control.[E] Over-expression of DmPI31 is sufficient for caspase activation in the absence ofnutcracker function. Active caspase-3 staining is shown in green and actin filaments (phalloidin staining) in red. Over-expression of DmPI31 in wild-type testes has no detectable effect on caspase-3 staining (left panel). Whereasnutcrackerms771−/− mutant testes lack caspase-3 staining (middle panel), expression of DmPI31 restores readily detectable caspase activity (right panel).[Table 1] Summary ofnutcracker mutant rescue experiments. nutcrackerms771−/− (ms771−/) phenotypes rescued by ectopic expression of either wild-typenutcracker (nutcracker-WT), anutcracker construct containing a mutation that prevents binding to DmPI31 (nutcrackerV-E), or a DmPI31 construct (PI31). A control for equal expression is shown in Figure S3B.
Figure 4
Figure 4. DmPI31 can stimulate proteasome activityin vitro
[A–B]In vitro proteasome activity assays using Suc-LLVY-AMC substrate. Rates are plotted relative to that of the control sample lacking DmPI31. Each experiment was repeated several times with similar results. Error bars represent the standard deviation from three independent readings.[A] DmPI31 is an inhibitor of 20S but an activator of the 26S proteasomes. Increasing concentrations of purified DmPI31 were used and the effect on the activity of purified bovine 20S or 26S proteasomes was monitored by rate of fluorogenic substrate hydrolysis. 0.15μg 20S or 0.05μg 26S were used per reaction (rxn).[B] DmPI31 lacking the HbYX motif displays reduced ability to activate mammalian 26S proteasomes. These experiments were preformed as in [A] with 26S proteasomes. The K(apparent) was calculated by using the concentration of DmPI31 or DmPI31-HbYX at saturation.
Figure 5
Figure 5. DmPI31 stimulates proteasome functionin vivo
Inhibition of proteasome activity in the developing retina causes phenotypes that are sensitive to the amounts of DmPI31 protein.[A-E] Effects of expressing DmPI31 at 29°C.[A] GMR-Gal4 expression in a wild type background causes no detectable phenotype.[B] Inhibition of proteasome function upon expression of the dominant-negative temperature-sensitive mutants UAS-DTS5 and UAS-DTS7 in the developing retina at 29°C causes a small rough eye phenotype.[C] This phenotype is not affected by co-expressing GFP, but[D] is suppressed by co-expressing full-length DmPI31, indicating that elevated levels of DmPI31 can boost proteasome activity.[E] Western blot analysis of total eye lysates. The suppression of the rough eye phenotype is not caused by upregulation of the proteasome subunits, as verified by probing alpha7 subunit levels.[F–N] Depletion of DmPI31 enhances the eye phenotypes caused by reduced proteasome activity. Compared to flies expressing eitherdmPI31RNAi[F] or the DTS proteasome mutants[G] at 25°C, combined expression of bothdmPI31RNAi and DTS proteasome mutant transgenes causes severe defects in eye development[H–I]. dmPI31RNAi enhances the eye phenotypes of both DTS7 and DTS5 at 25°C.[J] Western blot demonstrating that thedmPI31RNAi construct results in DmPI31 knock-down.[K-N] Expressing either DTS5 or DTS7 together withdmPI31RNAi at 29°C, but not each alone, results in lethality. Structure-function studies of dmPI31 are summarized in Table S1.
Figure 6
Figure 6. DmPI31 has an essential physiological function and is required for normal proteasome activityin vivo
[A–F]dmPI31 mutants are lethal, but transgenic expression of DmPI31 in the soma rescues this lethality and permits the recovery of adult flies that lack DmPI31 in germ cells. These flies display defects in the cell-cycle and protein degradation. The left panel depicts wild-type testes, while the right depictsdmPI31 mutants that have been rescued to adulthood.[A–A′] Distribution of DmPI31 protein in the testis. Testes were stained with and antibody staining towards DmPI31 (green). The wild type testis displays staining in germ-line stem cells (GSCs) and primary spermatocytes. This staining is lost indmPI31 mutant testes. The inset depicts a larger magnification of the apical tip, where these cells reside.[B–B′] BrdU incorporation assay to detect dividing GSCs. Anti-BrdU staining (green) labeling S-phase cells is detected at the apical tip of the wild type testis. A similar labeling is detected in thedmPI31 mutant testis, indicating that mitotic divisions are not disrupted. Nuclei are stained with DAPI (blue).[C–C′] Phospho-histone3 (PH3) antibody staining (green), which marks meiotic divisions. The PH3 antibody stains dividing nuclei, and therefore detects cells that are undergoing either mitosis or meiosis. Compared to wild-type testis, which display staining in the nuclei of 32 cell cysts, no staining is detected in cysts ofdmPI31 mutant testis, suggesting that meiosis is stunted. An arrow points to a 32-cell cyst (enlarged in inset) where PH3 staining is detected in wild type.[D–D′] Anti-Vasa antibody staining (green). This antibody is specific for germ cell progenitors. During normal differentiation, Vasa staining is strongest in GSCs and primary spermatocytes, and disappears when cells approach meiosis. In contrast,dmPI31 mutant testes contain cysts with persistent Vasa staining, indicating that these cells maintain progenitor identity and fail to differentiate.[E–E′] CyclinB staining. CyclinB is normally detected in 16 cell stage cysts and disappears just before meiosis completes. IndmPI31 mutant cells, CyclinB persists in 16-cell stage cysts, indicating that the degradation of this protein does not occur normally. See also Figure S4.[F–F′] FK2 staining of ubiquitin-conjugated proteins. FK2 detects the abundance of poly-ubiquitnated proteins and is thus a detector of proteasome activity. In normal germ-line cells (a pre-meiotic cyst is circled), FK2 staining is diffuse, and prominent staining is detected in differentiating nuclei (asterisks in separate cyst). In contrast, mostdmPI31 mutant cells display punctate staining (arrows point to poly-ubiquitnated protein clusters), indicating accumulation of non-degraded poly-ubiquitinated proteins.
Figure 7
Figure 7. Model for proteasome regulation by the Nutcracker-DmPI31 complex
During their terminal differentiation, spermatids undergo a severe reduction in cell volume. This process, termed “individualization”, requires proteasome activity (Zhong and Belote, 2007). Both Nutcracker and DmPI31 are required for normal proteasome activity in the testis (Bader et al., 2010). The binding of Nutcracker stabilizes DmPI31 by protecting the carboxy-terminal region of DmPI31 from cleavage. The C-terminal domain of DmPI31 is necessary for both binding to proteasomes and to stimulate their activity (McCutchen-Maloney et al., 2000). This domain contains a HbYX motif that has been implicated in proteasome gate opening (Smith et al., 2007). The formation and binding of the DmPI31-F-box regulatory complex stimulates proteasome activity and promotes caspase activation and spermatid differentiation. Since the function of Nutcracker is restricted to the testis, whereas DmPI31 has an essential function in somatic cells, it is likely that other factors contribute to the regulation of DmPI31.
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