Ubiquitin-dependent proteolysis can initiate at ribosomes for myriad reasons including misfolding of a nascent chain or stalling of the ribosome during translation of mRNA. Clearance of a stalled complex is required to recycle the ribosome for future use. Here we show that the ubiquitin (Ub) pathway segregase Cdc48/p97 and its adaptors Ufd1-Npl4 participate in ribosome-associated degradation (RAD) by mediating the clearance of ubiquitinated, tRNA-linked nascent peptides from ribosomes. Through characterization of both endogenously-generated and heterologous model substrates for the RAD pathway, we conclude that budding yeast Cdc48 functions downstream of the Ub ligases Ltn1 and Ubr1 to release nascent proteins from the ribosome so that they can be degraded by the proteasome. Defective RAD could contribute to the pathophysiology of human diseases caused by mutations in p97.
Ribosomes are complex molecular machines that translate the sequence of bases in a messenger RNA (mRNA) transcript into a polypeptide that subsequently folds to form a protein. Each ribosome is composed of two major subunits: the small subunit reads the mRNA transcript, and the large subunit joins amino acids together to form the polypeptide. This process stops when the ribosome encounters a stop codon and releases the completed polypeptide.
It is critical that cells perform some form of quality control on the polypeptides as they are translated to prevent a build up of incomplete, incorrect or toxic proteins in cells. Problems can occur if a ribosome stalls while translating the mRNA transcript, or if the mRNA transcript is defective. For example, most mRNA transcripts contain a stop codon, but some do not, and these non-stop mRNA transcripts result in a non-stop polypeptide that remains tethered to the ribosome. It is important that the cell identifies and removes these faulty polypeptides so as to leave the ribosome free to translate other (non-faulty) mRNA transcripts. A regulatory protein called ubiquitin is responsible for marking and sending proteins that are faulty, or are no longer needed by the cell, to a molecular machine called the proteasome, where they are degraded by a process called proteolysis. In 2010 researchers identified Ltn1 as the enzyme that attaches ubiquitin to non-stop proteins in yeast.
Now, building on this work, Verma et al. identify additional proteins involved in this process. In particular, an ATPase enzyme called Cdc48 (known as p97 or VCP in human cells) and two co-factors—Ufd1 and Npl4—promote release of the ubiquitinated non-stop polypeptides from the ribosomes, thus committing the marked polypeptide to destruction by the proteasome. Verma et al. also show that the Cdc48-Ufd1-Npl4 complex is involved in other aspects of quality control of newly synthesized proteins within cells. Collectively these processes are known as ribosome-associated degradation.
Mutations of the gene that codes for human p97 can cause a number of diseases, including Paget's disease of the bone and frontotemporal dementia, so an improved understanding of ribosome-associated degradation could provide new insights into these diseases.