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, 23 (7), 2754-73

The Defective Proteasome but Not Substrate Recognition Function Is Responsible for the Null Phenotypes of the Arabidopsis Proteasome Subunit RPN10

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The Defective Proteasome but Not Substrate Recognition Function Is Responsible for the Null Phenotypes of the Arabidopsis Proteasome Subunit RPN10

Ya-Ling Lin et al. Plant Cell.

Abstract

Ubiquitylated substrate recognition during ubiquitin/proteasome-mediated proteolysis (UPP) is mediated directly by the proteasome subunits RPN10 and RPN13 and indirectly by ubiquitin-like (UBL) and ubiquitin-associated (UBA) domain-containing factors. To dissect the complexity and functional roles of UPP substrate recognition in Arabidopsis thaliana, potential UPP substrate receptors were characterized. RPN10 and members of the UBL-UBA-containing RAD23 and DSK2 families displayed strong affinities for Lys-48-linked ubiquitin chains (the major UPP signals), indicating that they are involved in ubiquitylated substrate recognition. Additionally, RPN10 uses distinct interfaces as primary proteasomal docking sites for RAD23s and DSK2s. Analyses of T-DNA insertion knockout or RNA interference knockdown mutants of potential UPP ubiquitin receptors, including RPN10, RPN13, RAD23a-d, DSK2a-b, DDI1, and NUB1, demonstrated that only the RPN10 mutant gave clear phenotypes. The null rpn10-2 showed decreased double-capped proteasomes, increased 20S core complexes, and pleiotropic vegetative and reproductive growth phenotypes. Surprisingly, the observed rpn10-2 phenotypes were rescued by a RPN10 variant defective in substrate recognition, indicating that the defectiveness of RPN10 in proteasome but not substrate recognition function is responsible for the null phenotypes. Our results suggest that redundant recognition pathways likely are used in Arabidopsis to target ubiquitylated substrates for proteasomal degradation and that their specific roles in vivo require further examination.

Figures

Figure 1.
Figure 1.
Protein Expression and Phenotypes of Arabidopsis T-DNA or RNAi Lines for Loci Encoding Various UPP Ubiquitin Receptors. (A) The expression levels in rosette leaves or seedlings for the proteins encoded by genes targeted in various Arabidopsis T-DNA or RNAi lines, including rpn10-2; rpn13-1; rad23a-2, rad23b-3, rad23c-2, rad23d-1, rad23abc, rad23acd, rad23bcd, and rad23abcd (rad23s); dsk2a-1 and dsk2b-1 (dsk2s); dsk2-RNAi lines; ddi1-2 and ddi1-3 (ddi1s); and nub1-1, were determined by immunoblotting using antisera prepared separately with recombinant proteins, as indicated at the right. The expected mobilized positions of the various ubiquitin receptors are indicated with arrows, and the members’ alphabetical extensions are indicated when multiple members exist. The mobilized positions marked for RAD23a-d were determined using single, triple, and quadruple knockout mutants for four RAD23 gene members. The DDI1 and RPN10 expression levels or Brilliant Blue R (BBR)–stained gels are included to confirm that equal protein samples from Col-0 and insertion line(s) were loaded. For rpn13-1, the mobilized positions for protein size markers (in kilodaltons) are indicated at the left. The asterisks mark the positions of nonspecific proteins that were detected with the rpn13-1 protein sample. (B) Representative 7- (D7) and 14-d-old (D14) seedlings from various T-DNA or RNAi lines.
Figure 2.
Figure 2.
Reduced Plant Growth Rate and Delayed Flowering Time Are Associated with the rpn10-2 Line. (A) The expression levels of RPN10 and 20S proteasome subunits in rpn10-2 and the complementation lines. The expression of RPN10 (α-N10) and 20S proteasome subunits (α-20S) in 7-d-old seedlings from Col-0, rpn10-2, and complementation lines cN10 and u123 were examined by immunoblotting. The Brilliant Blue R (BBR)–stained duplicate gels were examined to confirm equal loading. The obvious mobility shift of the triple-UIM RPN10 mutant likely is due to the UIM1 mutation, as noted previously (Fu et al., 1998). (B) Representative 22, 45, and 76 DAS Col-0, rpn10-2, and complementation plants are shown. The reduced growth rate and increased final plant height associated with rpn10-2 plants were restored in the complementation lines. (C) The flowering time, recorded by DAS (left) or rosette leaf numbers (right), for Col-0, rpn10-2, and complementation lines. The flowering time was recorded when the floral stalk reached ~1 cm. The numbers indicated above the histograms are the sample sizes. The error bars represent the sd. **P < 0.01, rpn10-2 was compared with Col-0; ++P < 0.01, the complementation lines were compared with rpn10-2 using Student’s t test.
Figure 3.
Figure 3.
Leaf Morphology and Delayed Dark-Induced Leaf Senescence of rpn10-2. (A) Representative 45 DAS cauline and rosette leaves from Col-0, rpn10-2, and complementation plants are shown. (B) Representative cross sections of 28 DAS rosette leaves from Col-0 and rpn10-2 plants. (C) Rosette leaf thickness for Col-0, rpn10-2, c35S-1, and c35S-2 plants. Sample sizes (n) are indicated. The error bars represent the sd. **P < 0.01, rpn10-2 was compared with Col-0; ++P < 0.01, c35S-1 and c35S-2 were compared with rpn10-2 using Student’s t test. (D) The trichome branch number distribution on the upper epidermis of 55 DAS rosette leaves from Col-0, rpn10-2, and complementation plants. Derived from three to four plants, the numbers of trichomes observed with a Stemi SV6 stereomicroscope (Carl Zeiss) are indicated above the histograms. The classes of different branch numbers (2 to 6) are color-coded as indicated. (E) rpn10-2 displayed delayed dark-induced leaf senescence. Mature 28 DAS rosette leaves from Col-0, rpn10-2, cN10, and u123 plants were shown before (Day 0) and after 8 d (Day 8) incubation in the dark.
Figure 4.
Figure 4.
The rpn10-2 Plants Are Sterile and Have Defective Male and Female Gamete Function. (A) Representative 46 DAS siliques from Col-0, rpn10-2, and complementation plants are shown. (B) The average ovule numbers per carpel from Col-0, rpn10-2, and complementation plants. (C) The average percentages of aborted ovules from siliques of the Col-0, rpn10-2, and complementation plants. **P < 0.01, rpn10-2 was compared with Col-0; ++P < 0.01, the complementation lines were compared with rpn10-2 using Student’s t test in (B) and (C). (D) and (E) The male and female gamete function of rpn10-2 (n10) plants (D) or c35S-complemented rpn10-2 lines (c35S-1, c35S-2, and c35S-4) (E) was assessed by reciprocal crossing with Col-0 (Wt). The average percentages of aborted ovules in reciprocal crossed siliques are shown. Those listed first for all cross combinations are the female parents. The ovule abortion rate in manually self-crossed siliques of Col-0 (Wt x Wt) or rpn10-2 (n10 x n10) served as a control. **P < 0.01, compared with the wild-type crossing control (Wt x Wt) using Student’s t test. (F) The expression levels of RPN10, 20S proteasome subunits, DDI1, and CSN5 in pollen and flowers from Col-0, cN10-25, cN10-19, and c35S-2 plants were examined by immunoblotting (α-RPN10, α-20S, α-DDI1, and α-CSN5). CSN5 was examined as a loading control. (B) to (E) The numbers of siliques examined or crossed are indicated above the histograms, and the error bars are the sd.
Figure 5.
Figure 5.
Larger Anthers and Pollen and Distorted Developmental Stage Distribution of Female Gametes Are Observed with the rpn10-2 Flowers. (A) Representative anthers from stage 13 Col-0 and rpn10-2 flowers are shown using cryo-SEM. Note that the images are shown with different scales; the rpn10-2 plants have larger anthers than do the Col-0 plants. The red-boxed regions contain identical areas and are shown as close-ups in (B). (B) The representative rpn10-2 anther contains larger and shrunken pollen grains, marked by red arrowheads, compared with the typical wild-type Col-0 pollen grains. (C) The relative diameters of pollen grains from the Col-0, rpn10-2, and complementation plants. The pollen grains were visualized by Alexander’s stain, and the numbers of pollen grains measured (n) are indicated. The error bars represent the sd. **P < 0.01, rpn10-2 was compared with Col-0; ++P < 0.01, the complementation lines were compared with rpn10-2 using Student’s t test. (D) Representative anther cross-sections from stage 12 Col-0 and rpn10-2 flowers. Larger, irregular, and shrunken pollen are associated with rpn10-2 anthers, which often are full of small vacuoles. (E) DAPI-stained pollen from Col-0, rpn10-2, c35S-2, and cN10-25 plants. The typical trinucleate pattern is associated only with Col-0 and cN10-25 plants; it is not associated with rpn10-2 and c35S-2 plants. (F) The developmental stage distribution of the female gametophytes in flowers of different stages from Col-0 and rpn10-2 plants. The ovules were collected from Col-0 and rpn10-2 flowers at stage 12a, 12b, and 12c, from Col-0 flowers 2 d after emasculation (em) and from stage 13 rpn10-2 flowers. The ovule stage distributions are indicated as color-coded horizontal bars with numbers of observed ovules. The female gametophyte developmental stages were determined using CLSM and were classified as FG0-7, as previously described (Christensen et al., 1998). Large portions of the rpn10-2 female gametophytes from flowers of different stages contained only degenerated nuclei or lacked nuclei altogether (DG).
Figure 6.
Figure 6.
Significant Reproductive Growth Phenotypes Are Associated with the rpn10-2 Plants. (A) A comparison of representative 30 DAS Col-0 and rpn10-2 plants shows that the latter exhibit a reduction of axillary inflorescences. Red arrowheads mark the secondary inflorescences that are associated with the cauline leaves of the Col-0 plant; white arrowheads mark the cauline leaves of the rpn10-2 plant, in which secondary inflorescences are not visible. (B) Representative Col-0 and rpn10-2 plants show differential accumulation of anthocyanin on the surfaces of the top floral stems. (C) Representative flowers from Col-0, rpn10-2, and complementation plants. (D) Average pedicel lengths of siliques from Col-0, rpn10-2, and complementation plants. The numbers of siliques examined are indicated above the histograms. (E) Relative petal areas for Col-0, rpn10-2, and complementation flowers. The average area of the Col-0 petals was set to 1. The sample sizes are indicated on the top of the histograms. (F) A comparison of petal adaxial epidermal cells from Col-0 and rpn10-2 flowers using cryo-SEM demonstrates that the latter are markedly larger in size. (G) Relative cell size of the adaxial petal epidermis from the Col-0, rpn10-2, and c35S flowers. The average cell size of the Col-0 petals was set to 1. The relative cell size for each line was derived from 9 to 11 area units (0.082 mm2/unit) as indicated using cryo-SEM. (H) The stamen heights of Col-0, rpn10-2, and complementation plants. The examined numbers of stamens, collected from 10 flowers, are indicated. (I) The anther heights and widths of Col-0, rpn10-2, and complementation plants. The examined numbers of anthers, collected from 10 flowers, are indicated. (J) The pistil heights and widths of Col-0, rpn10-2, and complementation plants. The numbers of pistils examined are indicated. The error bars are the sd. **P < 0.01, rpn10-2 was compared with Col-0; ++P < 0.01, the complementation lines were compared with rpn10-2 using Student’s t test in (D), (E), and (G) to (J).
Figure 7.
Figure 7.
Feedback Regulation of Ubiquitin Receptors and Altered Proteasome Complex Abundance in rpn10-2 Plants. (A) The expression of various base and lid subunits, 20S proteasome subunits, and UBL-UBA factors in the rosette leaves of rpn10-2 and the complementation plants. The expression of RPN10, RPN13, RPT5, RPN8, 20S proteasome subunits, RAD23a-d, DSK2a-b, and DDI1 in 30 DAS rosette leaves from Col-0, rpn10-2, cN10-19, and c35S-2 was examined by immunoblotting. CSN5 expression was included as a loading control. The various antisera used were prepared separately against recombinant Arabidopsis RPN10, RPN13, RPT5a, RPN8a, RAD23b, RAD23c, DSK2b, DDI1, and CSN5 and against purified moss 20S proteasome (α-20S). The mobilized positions, as marked by arrows for RAD23a (a), RAD23bi (bi), RAD23bii (bii), and RAD23d (d), were determined using single, triple, and quadruple knockout mutants for the four RAD23 gene members (Figure 1A). Family members for RPT5, RPN8, and DSK2 were not resolved and are also marked with arrows (a and b). The proteasome subunits shown are the prominent signals detected with the moss 20S proteasome antisera; their specific identities have not been determined. (B) Protein expression in the rosette leaves of rpn10-2 expressing the triple UIM mutated RPN10 variant. The expression of RPN10, 20S proteasome subunits, RAD23a/bii, RAD23d, and DDI1 in 30 DAS rosette leaves from Col-0, rpn10-2, and the cN10 and u123 complementation lines was examined by immunoblotting using the indicated antisera. CSN5 expression was included as a loading control. (C) and (D) The relative abundances of core (CP), single-capped (RP1-CP), and double-capped (RP2-CP) proteasomes in Col-0, rpn10-2, cN10-19, and c35S-2 rosette leaves were examined by native PAGE in conjunction with immunoblotting using antisera against RPN10, RPT5a, RPN6, RPN8a, or the 20S proteasome (α-20S) (C) or with in-gel activity assays in the presence or absence of 0.02% SDS (D). The arrowheads mark the positions of the various proteasome complexes. Equal amounts of protein (50 to 200 μg) from different samples were analyzed. The slight mobility variation in different blots for major complexes is due to different PAGE run times. (E) The relative abundances of core (CP), single-capped (RP1-CP), and double-capped (RP2-CP) proteasomes in Col-0, rpn10-2, cN10, and u123 rosette leaves were examined by native PAGE in conjunction with immunoblotting using antisera against the 20S proteasome (α-20S) (top) or with in-gel activity assays in the presence or absence of 0.02% SDS (bottom). The arrowheads mark the positions of the various proteasome complexes.

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