Concurrent Translocation of Multiple Polypeptide Chains through the Proteasomal Degradation Channel

Abstract

The proteasome can actively unfold proteins by sequentially unraveling their substrates from the attachment point of the degradation signal. To investigate the steric constraints imposed on substrate proteins during their degradation by the proteasome, we constructed a model protein in which specific parts of the polypeptide chain were covalently connected through disulfide bridges. The cross-linked model proteins were fully degraded by the proteasome, but two or more cross-links retarded the degradation slightly. These results suggest that the pore of the proteasome allows the concurrent passage of at least three stretches of a polypeptide chain. A degradation channel that can tolerate some steric bulk may reconcile the two opposing needs for degradation that is compartmentalized to avoid aberrant proteolysis yet able to handle a range of substrates of various sizes.Protein degradation is a critical part of cellular regulation (1). In eukaryotic cells, a multicomponent protease called the proteasome is responsible for the turnover of short-lived regulatory proteins, the removal of abnormal polypeptides, and the production of peptides for antigen presentation (2). Degradation of short-lived regulatory proteins is essential for a wide range of cellular functions including cell cycle control and signal transduction (3). Failure to degrade aggregates of misfolded proteins can lead to disease such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease (4). Degradation by the proteasome usually involves two consecutive steps: targeting of the substrate for degradation by the attachment of polyubiquitin chains and degradation of the tagged protein by the proteasome with concomitant release of ubiquitin (5).The active sites of proteolysis of the proteasome by themselves show little substrate specificity. Specificity of degradation is achieved by sequestering the proteolytic sites within the structure and tightly controlling access. The three-dimensional structure of the proteasome has been determined by x-ray crystallography and electron microscopy (6 -11). The proteasome consists of a central proteolytic core particle with regulatory caps at either end of it. The core particle is made of two copies of seven different ␣-subunits and seven different ␤-subunits. The subunits are arranged in four heptameric rings, which are stacked on top of each other to form a cylindrical particle. The rings form a central ␤-chamber and two ␣-chambers, one at each end of the particle (2). The proteolytic sites are located in the ␤-chamber and are accessible only through a central channel that runs along the long axis of the particle. The channel has narrow constrictions at the entrance and exit of the ␣-chamber (2). In the isolated yeast core particle, the entrance to the degradation channel is blocked by the N termini of the ␣-subunits (7). The channel opens when the regulatory caps bind to the core (12). The caps consist of 17 subunits, six of which have ATPase activity, and contact the co...

The E3 Ubiquitin Ligase UBE3C Enhances Proteasome Processivity by Ubiquitinating Partially Proteolyzed Substrates

“…4B). However, it has been reported that the proteasome is able to concurrently degrade multiple polypeptide chains, so the presence of a stalled fragment may not inhibit degradation of other proteins (60). Because overexpression of UBE3C does not accelerate degradation (Fig.…”

“…Surprisingly, similar treatment with proteasome inhibitor did not yield results similar to treatment with 17-AAG. Treatment with either drug is known to cause a heat shock response in cells (20,60). Induction of this response may be sufficient to compensate for the loss of UBE3C activity in the case of proteasome inhibition.…”

Splicing of Distant Peptide Fragments Occurs in the Proteasome by Transpeptidation and Produces the Spliced Antigenic Peptide Derived from Fibroblast Growth Factor-5

“…Such trans-splicing is conceivable in theory, because it was shown that the inner channel of the proteasome, with a diameter of 13 Å (17), can accommodate more than one polypeptide chain (18,19). The proteasome was also able to bind and process two substrates simultaneously: one in each catalytic chamber (20).…”

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)

“…We propose that the MIM-MIT interaction serves to recruit Vps4 to ESCRT-III polymers and to unmask or activate the binding site at the hexamer pore, which possesses an inherent ability to bind and translocate a wide variety of sequences starting at an internal loop, such as has been described for the proteasome (69,70). An attractive feature of this model is that it provides a mechanism for avoiding Vps4 pore engagement of inappropriate substrates.…”