Dissection of the Carboxyl-Terminal Domain of the Proteasomal Subunit Rpn11 in Maintenance of Mitochondrial Structure and Function

Abstract

We have previously demonstrated that the C-terminal part of Rpn11, a deubiquitinating enzyme in the lid of the proteasome, is essential for maintaining a correct cell cycle and normal mitochondrial morphology and function. The two roles are apparently unlinked as the mitochondrial role is mapped to the Carboxy-terminus, whereas the catalytic deubiquitinating activity is found within the N-terminal region. The mitochondrial defects are observed in rpn11-m1 (originally termed mpr1-1), a mutation that generates Rpn11 lacking the last 31 amino acids. No mitochondrial phenotypes are recorded for mutations in the MPN+/JAMM motif. In the present study, we investigated the participation of the last 31 amino acids of the Rpn11 protein by analysis of intragenic revertants and site-specific mutants. We identified a putative alpha-helix necessary for the maintenance of a correct cell cycle and determined that a very short region at the C-terminus of Rpn11 is essential for the maintenance of tubular mitochondrial morphology. Furthermore, we show that expression of the C-terminal part of Rpn11 is able to complement in trans all of the rpn11-m1 mitochondrial phenotypes. Finally, we investigate the mechanisms by which Rpn11 controls the mitochondrial shape and show that Rpn11 may regulate the mitochondrial fission and tubulation processes.

Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

“…Expression of the carboxyl terminus of Rpn11 (GST-C Rpn11 ) 2 partially suppressed the diverse defects of rpn11-1, consistent with other studies that showed the this domain is required for Rpn11 function (21)(22)(23). We found that the high levels of multiubiquitinated proteins in rpn11-1 were bound to shuttle factors.…”

“…Direct binding studies failed to reveal any interaction between FLAG-rpn11-1 and GST-C Rpn11 , indicating that suppression is not achieved by dimerization of these protein sequences (data not shown and Ref. 22). Because rpn11-1 protein is catalytically active but destabilizes proteasomes, we propose that the carboxyl terminus promotes proteasome stability, consistent with previous reports showing its requirement for Rpn11 function (21)(22)(23).…”

“…22). Because rpn11-1 protein is catalytically active but destabilizes proteasomes, we propose that the carboxyl terminus promotes proteasome stability, consistent with previous reports showing its requirement for Rpn11 function (21)(22)(23).…”

“…We investigated whether the expression of this domain in trans could alleviate the growth and proteolytic defects of rpn11-1. A recent study showed that the C-terminal one-third of Rpn11 could overcome the mitochondrial and DNA repair defects of rpn11-1 (22) but not its proteolytic deficiency. We expressed the carboxyl-terminal 39 residues of rpn11-1 (GST-C Rpn11 ) in RPN11 and rpn11-1 (Fig.…”

The toxicity of an “artificial” amyloid is related to how it interacts with membranes

“…It is derived from pYEF1mtRFP, 44 which was constructed using PYX-mtGFP. 45 The selectable marker was changed from URA3 (pYEF1mtRFP) to LEU2 (pYEF2mtRFP).…”