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The ubiquitin-proteasome system is responsible for the regulation and turnover of many short-lived proteins both in the cytoplasm and in the nucleus. Degradationcanoccurviatwodistinctpathways,anNterminusdependent pathway and a lysine-dependent pathway. The pathways are characterized by the site of initial ubiquitination of the protein, the N terminus or an internal lysine, respectively. MyoD, a basic helix-loop-helix transcription factor, is a substrate for the ubiquitinproteasome pathway and is degraded in the nucleus. It is preferentially tagged for degradation on the N terminus and thus is degraded by the N terminus-dependent pathway. Addition of a 6؋ Myc tag to the N terminus of MyoD can force degradation through the lysine-dependent pathway by preventing ubiquitination at the N-terminal site. Modifications of the nuclear localization signal and nuclear export signal of MyoD restrict ubiquitination and degradation to the cytoplasm or the nucleus. Using these mutants, we determined which degradation pathway is dominant in the cytoplasm and the nucleus. Our results suggest that the lysine-dependent pathway is the more active pathway within the cytoplasm, whereas in the nucleus the two pathways are both active in protein degradation.Degradation of many short-lived cellular proteins, such as transcription factors, tumor suppressors, and cell cycle regulators, occurs via the ubiquitin-proteasome pathway (1-4). Through this pathway, proteins are targeted for degradation by the 26 S proteasome via the formation of a polyubiquitin chain. The process begins with activation of ubiquitin by the ubiquitin-activating enzyme (E1), 1 followed by transfer of ubiquitin to E2, a ubiquitin-conjugating enzyme. E2 shuttles the ubiquitin molecule to the substrate-specific ubiquitin ligase (E3), which then delivers the ubiquitin to the substrate to be degraded. Initially, it was thought that ubiquitination occurred only through a lysine-dependent ubiquitination pathway in which ubiquitin is covalently attached to the substrate protein via an amide linkage to the ⑀-amino group of an internal lysine (5). However, recent studies have shown that the N terminus of a protein substrate may also serve as the site of ubiquitination (6 -8), a pathway termed N terminus-dependent ubiquitination. Via either ubiquitination pathway, polyubiquitin chain formation continues by the conjugation of subsequent ubiquitin moieties to the attached ubiquitin, and the substrate-ubiquitin conjugate is then degraded by the 26 S proteasome in an ATP-dependent manner. Isopeptidases cleave the ubiquitin chain, and the single ubiquitin molecules are recycled (5). Currently, the relative contribution of each of these two pathways is unknown.Among the short-lived proteins degraded by the ubiquitinproteasome system are several transcription factors, including MyoD. MyoD, a nuclear basic helix-loop-helix transcription factor necessary for skeletal muscle differentiation (9), is rapidly degraded by the ubiquitin-proteasome system both in vitro and in vivo (10). Ubiqu...

Human XPA is an essential component in the multienzyme nucleotide excision repair (NER) pathway. The solution structure of the minimal DNA binding domain of XPA (XPA-MBD: M98-F219) was recently determined [Buchko et al. (1998) Nucleic Acids Res. 26, 2779-2788, Ikegami et al. (1998) Nat. Struct. Biol. 5, 701-706] and shown to consist of a compact zinc-binding core and a loop-rich C-terminal subdomain connected by a linker sequence. Here, the solution structure of XPA-MBD was further refined using an entirely new class of restraints based on pseudocontact shifts measured in cobalt-substituted XPA-MBD. Using this structure, the surface of XPA-MBD which interacts with DNA and a fragment of the largest subunit of replication protein A (RPA70 Delta C327: M1-Y326) was determined using chemical shift mapping. DNA binding in XPA-MBD was highly localized in the loop-rich subdomain for DNA with or without a lesion [dihydrothymidine (dhT) or 6-4-thymidine-cytidine (64TC)], or with DNA in single- or double-stranded form, indicating that the character of the lesion itself is not the driving force for XPA binding DNA. RPA70 Delta C327 was found to contact regions in both the zinc-binding and loop-rich subdomains. Some overlap of the DNA and RPA70 Delta C327 binding regions was observed in the loop-rich subdomain, indicating a possible cooperative DNA-binding mode between XPA and RPA70 Delta C327. To complement the chemical shift mapping data, the backbone dynamics of free XPA-MBD and XPA-MBD bound to DNA oligomers containing dhT or 64TC lesions were investigated using 15N NMR relaxation data. The dynamic analyses for the XPA-MBD complexes with DNA revealed localized increases and decreases in S2 and an increase in the global correlation time. Regions of XPA-MBD with the largest increases in S2 overlapped regions having the largest chemical shifts changes upon binding DNA, indicating that the loop-rich subdomain becomes more rigid upon binding DNA. Interestingly, S2 decreased for some residues in the zinc-binding core upon DNA association, indicating a possible concerted structural rearrangement on binding DNA.