The mechanism underlying the delivery of ubiquitylated substrates to the proteasome is poorly understood. Rad23 is a putative adaptor molecule for this process because it interacts with ubiquitin chains through its ubiquitin-associated motifs (UBA) and with the proteasome through a ubiquitin-like element (UBL). Here, we demonstrate that the UBL motif of Rad23 also binds Ufd2, an E4 enzyme essential for ubiquitin chain assembly onto its substrates. Mutations in the UBL of Rad23 alter its interactions with Ufd2 and the proteasome, and impair its function in the UFD proteolytic pathway. Furthermore, Ufd2 and the proteasome subunit Rpn1 compete for the binding of Rad23, suggesting that Rad23 forms separate complexes with them. Importantly, we also find that the ability of other UBL/UBA proteins to associate with Ufd2 correlates with their differential involvement in the UFD pathway, suggesting that UBL-mediated interactions may contribute to the substrate specificity of these adaptors. We propose that the UBL motif, a protein-protein interaction module, may be used to facilitate coupling between substrate ubiquitylation and delivery, and to ensure the orderly handoff of the substrate from the ubiquitylation machinery to the proteasome.

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1–Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.

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Determining the half-life of proteins is critical for an understanding of virtually all cellular processes. Current methods for measuring in vivo protein stability, including large-scale approaches, are limited in their throughput or in their ability to discriminate among small differences in stability. We developed a new method, Stable-seq, which uses a simple genetic selection combined with highthroughput DNA sequencing to assess the in vivo stability of a large number of variants of a protein The regulation of protein stability is critical in order for cells to maintain proper functioning of almost every process. Thus, approaches for measuring the in vivo stability of a protein are essential for the identification of components in proteolytic pathways that affect protein turnover and for an understanding of the consequences of their activities. These approaches include traditional biochemical methods such as the Western blot, in which samples taken from time points after the inhibition of protein expression are fractionated via gel electrophoresis and the relevant protein is visualized with the use of an antibody. Another method allows one to track the degradation rate of newly synthesized proteins by metabolically labeling proteins with a radioisotope and following their radioactivity. A third method fuses a protein to a reporter enzyme like ␤-galactosidase, allowing the steady-state level of a protein to be measured via the enzymatic activity of the reporter enzyme. However, these small-scale methods are limited in the number of samples that they can analyze.Large-scale methods have been developed that allow the simultaneous quantitation of the in vivo stability of many proteins. For example, Yen et al. fused ϳ8,000 human proteins to green fluorescent protein (GFP) 1 and followed the amount of each protein over time by using fluorescence-activated cell sorting (FACS) (1). To identify proteins, the plasmids encoding the GFP fusions were isolated and PCR products derived from these plasmids were hybridized to a DNA microarray. This method was applied to identify the substrates of a ubiquitin ligase complex (2). However, this method is limited by the number of bins into which protein fusions can be sorted in the FACS analysis and, consequently, how fine changes in stability can be discriminated. Alternatively, quantitative mass spectrometry has been used to analyze the stability of native proteins (3), but this approach often requires costly labeling and extensive data analysis. Moreover, these large-scale methods generally cannot distinguish differences in in vivo stability, which are sometimes significant, that result from small changes in a protein, such as single amino acid substitutions.We present a method, Stable-seq, for measuring the in vivo stability of large numbers of variants of a protein that combines a simple genetic selection with high-throughput DNA sequencing. Stable-seq is a form of deep mutational scanning (4, 5), in which a physical association between each protein variant and the DNA that ...