Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

Abhishek Chandra,Li Chen,Huiyan Liang,Kiran Madura

doi:10.1074/jbc.m109.076786

Abstract

A major fraction of intracellular protein degradation is mediated by the proteasome. Successful degradation of these substrates requires ubiquitination and delivery to the proteasome followed by protein unfolding and disassembly of the multiubiquitin chain. Enzymes, such as Rpn11, dismantle multiubiquitin chains, and mutations can affect proteasome assembly and activity. We report that different rpn11 mutations can affect proteasome interaction with ubiquitinated proteins. Moreover, proteasomes are unstable in rpn11-1 and do not form productive interactions with multiubiquitinated proteins despite high levels in cell extracts. However, increased levels of ubiquitinated proteins were found associated with shuttle factors. In contrast to rpn11-1, proteasomes expressing a catalytically inactive mutant (rpn11(AXA)) were more stable and bound very high amounts of ubiquitinated substrates. Expression of the carboxyl-terminal domain of Rpn11 partially suppressed the growth and proteasome stability defects of rpn11-1. These results indicate that ubiquitinated substrates are preferentially delivered to intact proteasome.

Highlights

A major fraction of intracellular protein degradation is mediated by the proteasome, which consists of a catalytic (20 S) complex that is bound to two regulatory (19 S) particles [1]
Similar levels of Pre1-FLAG were recovered from each strain, the amount of 19 S subunits co-purified from rpn11-1 was significantly lower than from RPN11 and rpn11AXA
Nonspecific precipitation was not detected in a strain lacking Pre1FLAG (Ctrl, lane 1)

Summary

EXPERIMENTAL PROCEDURES

Plasmid DNA for generating an integrated derivative of Pre1-FLAG was provided by Dr J. DNA was partially digested with BsmI and transformed into the strains indicated in supplemental Table 1. (Plasmids are indicated in supplemental Table 2.) A plasmid for integrating FLAG-Rpn was prepared by amplifying the gene with its native promoter and cloning into Yiplac22 [24]. Plasmids expressing Rad, Ddi, and Dsk with an amino-terminal FLAG epitope were generated by PCR and cloned into Yeplac112. These genes were expressed from a PCUP1 promoter by the addition of 100 ␮M CuSO4. All the amplified DNAs were verified by sequencing both strands

Substrate Delivery to Proteasomes

RESULTS

Substrate Shuttle Factors Show

DISCUSSION