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1.
Figure 3

Figure 3. Mitochondrial structure in mpr1-1 and suppressed strains. From: Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain.

mpr1-1 was transformed with the constructs described in Figure , grown at 28 °C to the mid-exponential phase, stained with DASPMI, and mitochondria were observed by fluorescence microscopy. A WT-like mitochondrial tubular network is visualized in mpr1-1 only upon transformation with plasmids bearing either the WT RPN11 (B) or the CHR8-R11 chimaera (E).

Teresa Rinaldi, et al. Biochem J. 2004 July 1;381(Pt 1):275-285.
2.
Figure 6

Figure 6. Chimaeric proteins are incorporated into the proteasome and are present in non-associated forms. From: Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain.

Glycerol-gradient fractionation of yeast whole-cell extract was analysed for proteasome activity and the Rpn11 migration pattern. Peptidase activity indicates migration of WT proteasome (top panel). Fractions were also immunoblotted with anti-GFP for detecting the relevant constructs. Our antibodies were not sensitive enough to detect naturally abundant GFP-tagged Rpn11 constructs; however, upon overexpression of Rpn11–GFP, it can be found both in proteasome-associated and free (low-molecular-mass) fractions. Similarly, CHR8-R11-GFP expressed from high-copy plasmids is found incorporated into proteasomes, as well as in low-molecular-mass fractions. Migration of proteasomes in mpr1-1 that were not covered with an Rpn11-expressing construct was determined with anti-Rpt1. A slight shift towards lower-molecular-mass fractions is observed for proteasomes in mpr1-1, which is corrected for upon expression of Rpn11 or Rpn11 chimaeric proteins (see also Figure ).

Teresa Rinaldi, et al. Biochem J. 2004 July 1;381(Pt 1):275-285.
3.
Figure 8

Figure 8. RPN8 can suppress general proteolysis defects associated with mpr1-1. From: Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain.

(A) Accumulation of polyubiquitin (polyUb) conjugates in mpr1-1 at the permissive temperature can be rescued by overexpression of RPN8. Naturally abundant rpn11D (containing the WT C-terminus, but not a functional MPN+ motif) can also rescue polyubiquitin-conjugate accumulation, but to a lesser extent than multicopy RPN8. (B) Same as (A), after a 5 h shift to the restrictive temperature for mpr1-1. As expected, due to the heat-shock damage, polyubiquitinated conjugates accumulate even in WT to a greater extent than at the lower temperature. Accumulation in mpr1-1 is especially pronounced, but can be suppressed to a great extent by overexpression of RPN8 or naturally abundant rpn11D. The rpn11D mutant alone also accumulates polyubiquitinated substrates (right panel); however, this accumulation is not additive with accumulation due to the mpr1-1 mutation (left previously). The bottom panels (marked with an asterisk) indicate an equal protein loading in the lanes, and the positions of molecular-mass markers are shown on the left.

Teresa Rinaldi, et al. Biochem J. 2004 July 1;381(Pt 1):275-285.
4.
Figure 1

Figure 1. Overexpression of the RPN8 gene suppresses the cell cycle defect of the mpr1-1 mutant. From: Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain.

(A)Temperature-sensitivity of mpr1-1. The mpr1-1 mutant transformed with the [pYE-ScRPN8] plasmid (right panel) and mpr1-1 (transformed with an empty plasmid for comparison; left panel) were grown in glucose medium at 28 °C to the exponential-growth phase, and then shifted for 5 h to the non-permissive temperature (34 °C) and stained with DAPI. Pictures were taken from [rho °] cells to better appreciate the undivided nucleus of the mpr1-1 strain that migrates into the daughter cell. (B) Rounded mitochondria in mpr1-1 at the permissive temperature. Congenic WT-CMY, mpr1-1 and mpr1-1 harbouring [pYE-ScRPN8] were transformed with the pYX232-mtGFP plasmid expressing the GFP gene with a mitochondrial import sequence. Cells were cultured on glucose medium at 28 °C to the exponential-growth phase, harvested, stained with DAPI and observed by fluorescence microscopy.

Teresa Rinaldi, et al. Biochem J. 2004 July 1;381(Pt 1):275-285.
5.
Figure 4

Figure 4. Intragene complementation of mpr1-1 and rpn11 MPN mutations. From: Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain.

Suppression of the mitochondrial defect of mpr1-1 by plasmids expressing single amino acid substitutions in the MPN+ motif of Rpn11. (A) The mpr1-1 mutant was transformed with the following plasmids (counter-clockwise from the top right): an empty plasmid, WT RPN11 (pYC-RPN11), rpn11C116A (pYC-rpn11C), rpn11H111A (pYC-rpn11H), rpn11S119A (pYC-rpn11S) and rpn11D122A (pYC-rpn11D). Plates were incubated at the non-permissive temperature (34 °C) for 2 days. Independently, both mpn mutants and mpr1-1 are thermosensitive and lethal at 34 °C []. It should be noted that of the mpn mutations, rpn11H111A is the most severe []; therefore, unsurprisingly. it is slightly less efficient in rescuing mpr1-1, although it too rescues temperature sensitivity (left panel) and allows for growth on glycerol (right panel). (B) Normal tubular mitochondrial network in mpn mutants. The rpn11 point mutants were cultured to the mid-exponential growth phase at 28 °C and stained with DASPMI for visualization of mitochondria. The rpn11 point mpn mutants exhibit normal mitochondrial morphology.

Teresa Rinaldi, et al. Biochem J. 2004 July 1;381(Pt 1):275-285.
6.
Figure 2

Figure 2. Suppression of mpr1-1 phenotypes by Rpn8, and Rpn11-Rpn8 chimaeras. From: Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain.

(A) Schematic representation of various constructs used to study the mpr1-1 mutant. The MPN domain depicted in black covers most of the N-terminal region of Rpn11 and Rpn8. The MPN+ motif embedded within the MPN domain of Rpn11 is indicated by a large box, with the key residues highlighted in bold, and by underlining. Rpn11, mpr1-1 and the CHR11-R8 chimaera all contain this MPN+ motif. The C-terminal regions of Rpn11 and Rpn8 diverge significantly, and are shown either in white or by grey stripes respectively. The missense mutation in mpr1-1 leads to a truncation of residues 277–285, highlighted by the grey-shaded box. Numbers indicate the total amino acids in each construct. (B) Growth phenotypes: mpr1-1 was transformed with an empty plasmid (1 in the pie chart), pYE-RPN11 (2), pYE-RPN8 (3), pYC-RPN8 (4), pYE-CHR8-R11 (5), pYC-CHR8-R11 (6), pYE-CHR11-R8 (7) or pYE-HsRPN8 (8). Transformed strains were grown at 28 °C, streaked on to YPD or YPG plates and grown at 34 °C for 3 additional days. As mentioned in the text, constructs 3–8 alone do not rescue an rpn11 null, while construct 2 rescues the null creating a WT strain.

Teresa Rinaldi, et al. Biochem J. 2004 July 1;381(Pt 1):275-285.
7.
Figure 7

Figure 7. Proteasome structural defects in mpr1-1 are temperature-sensitive and can be suppressed by RPN8. From: Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain.

(A) Cell extracts were prepared from exponentially growing WT or mpr1-1 strains, or from other mutant strains that were brought to an identical cell density at the permissive temperature of 26 °C for mpr1-1. Extracts were clarified by centrifugation, and samples containing identical quantities of total protein were separated by non-denaturing PAGE (native gels). Proteasomes were visualized by the fluorogenic peptide overlay assay. Proteasome holoenzymes in WT are found as a mixture of symmetric doubly capped (RP2CP) and asymmetric singly capped (RP1CP) conformations. 26 S proteasomes in mpr1-1, however, are found almost exclusively in lidless forms, whether singly or doubly capped. Slightly higher levels of dissociated free 20 S CP are also evident. 20 S CP is easily visualized upon activation of the CP by SDS (right panel). (B) At 30 °C, dissociation of mpr1-1 is greatly pronounced. Almost no doubly capped form is present, and levels of singly capped base–CP complex are diminished. This structural defect can be suppressed to a great extent by overexpression of RPN8, and to a lesser degree by CHR8-R11. As a control, expression of WT RPN11 fully rescues the mpr1-1 proteasome structural defect.

Teresa Rinaldi, et al. Biochem J. 2004 July 1;381(Pt 1):275-285.
8.
Figure 5

Figure 5. Comparative growth phenotypes and stress response of mpr1-1 and MPN+ mutants of rpn11. From: Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain.

(A) Canavanine-sensitivity of WT-CMY, WT-BY and the congenic mutants mpr1-1 and rpn11D respectively. The same number of cells were spotted on to a YPD plate and on a minimal medium plate supplied with the required amino acids supplemented with 3 μg/ml of canavanine. (B) UV-sensitivity of WT-CMY (▴), WT-BY (•), the mpr1-1 mutant (*) and the point mutant rpn11D (♦). The strains were grown at a concentration of 2×107 cell/ml, plated on to YPD medium and irradiated at 0, 30, 60, 90 and 120 J/m2 (200 cells/plate; three plates for each point). Plates were incubated at 28 °C in the dark for 3 days and colonies were counted and averaged to determine residual viability compared with untreated cells. Comparisons should be made between each mutant and its congenic WT (see the Experimental section). (C) CHX=sensitivity of WT-CMY, WT-BY, the mpr1-1 mutant and the point mutant rpn11D. Serial dilutions of starting culture containing the same number of cells were spotted on to the YPD plate or on to YPD plates containing 0.05, 0.1 or 0.5 μg/ml CHX. (D) MMS-sensitivity of the mpr1-1 mutant transformed with the empty plasmid for control, or with pYC-RPN11, pYE-RPN8 or pYC-rpn11D plasmids, as well as the MMS-sensitivity of the rpn11D mutant strain and its congenic WT strain WT-BY. Cultures containing identical number of cells were spotted at 10-fold dilutions on to YPD medium or on to YPD medium containing 0.03% MMS.

Teresa Rinaldi, et al. Biochem J. 2004 July 1;381(Pt 1):275-285.

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