Cells were harvested at 8 or 24 h p.i. and lysed by freezing and thawing for three times. After centrifugation at 12 000 r.p.m. for 10 min, the supernatant was used in a CAT assay as described previously (Lin et al., 1996) . A Method to Identify p62's UBA Domain Interacting Proteins The UBA domain is a conserved sequence motif among polyubiquitin binding proteins. For the first time, we demonstrate a systematic, high throughput approach to identification of UBA domain-interacting proteins from a proteome-wide perspective. Using the rabbit reticulocyte lysate in vitro expression cloning system, we have successfully identified eleven proteins that interact with p62’s UBA domain, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer’s disease. Therefore, p62 may play a novel regulatory role through its UBA domain. Our approach provides an easy route to the characterization of UBA domain interacting proteins and its application will unfold the important roles that the UBA domain plays. p62 is a novel cellular protein which was initially identified in humans as a phosphotyrosine independent ligand of the src homology 2 (SH2) domain of p56 lck (1, 2) . p56 lck is a member of the c-src family of cytoplasmic tyrosine kinases that is found predominantly in cells of lymphoid origin (3, 4) . In addition to the interaction with p56 lck , p62 also associates with the Ser/Thr kinase (1, 2) , atypical protein kinase C (5, 6) , and ubiquitin (7) . In addition to the SH2 domain, p62 possesses several structural motifs, including a ubiquitin associated (UBA) domain that is capable of binding ubiquitin nonconvalently (8, 9) . Ubiquitin (Ub) is a small polypeptide of 76 amino acids that can be convalently attached to other proteins at specific lysine residues through chains composed of one (mono) or several ubiquitin moieties (poly). In addition to its classical role in protein degradation, ubiquitin is emerging as a signal for protein transport and processing (10) (11) (12) . Conjugation of ubiquitin to substrate proteins requires three enzymes: a ubiquitin activating enzyme E1, a ubiquitin-conjugating enzyme E2, and a ubiquitin ligase E3. Initially, E1 activates ubiquitin by forming a high energy thioester intermediate with the C-terminal glycine using ATP. The activated ubiquitin is sequentially transferred to E2, then to E3 which catalyzes isopeptide bond formation between the activated C-terminal glycine of ubiquitin and ε-amino group of a lysine residue of the substrate. Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin are attached to lysine side chains of the previously conjugated moiety to form branched polyubiquitin chains. The fate of ubiquitinated substrates depends on the number of ubiquitin moieties conjugated, as well as, the lysine linkage of Ub-Ub conjugation. The conjugation of ubiquitin to eukaryotic intracellular proteins is one way in which those proteins are targeted to the proteasome for subsequent rapid degradation. This mechanism is particularly important for short-lived regulatory proteins such as cyclins, cyclin-dependent protein kinase-inhibitors, p53, the nuclear factor kappa B precursor, and IκB (13) . The ubiquitinproteasome system consists of two steps: 1) the target protein is conjugated with polyubiquitin molecules, which mark the substrate for degradation; 2) the target protein is transferred to the 26S proteasome, unfolded and degraded.