Identification of an E1-like APP–BP1/hUba3 heterodimer for activation of NEDD8. (A) Sequence alignment of two peptides of the rabbit 62-kD protein associated with GST–NEDD8 (band 1 in Fig. 1B) with human APP–BP1 (Chow et al. 1996). Partial amino acid sequences of the fragments of the 62-kD band digested with lysylendopeptidase were determined with a protein sequencer and are shown above the APP–BP1 sequences in italics. (X) An unidentified residue. The identical amino acids are boxed in black. (B) Primary structure of human Uba3 (Hs) deduced from the nucleotide sequence of a human cDNA and its sequence alignment with yeast Uba3p (Sc) (PIR accession no. S54087). Amino acid residues are numbered from the amino terminus. Identical amino acids are boxed in black. The motif shown (•) is the consensus sequence for a nucleotide binding site. The asterisk indicates the essential Cys residue conserved in E1 enzymes that becomes linked to Ub in an E1–Ub thioester linkage. (C) Thioester linkage between ³⁵S-labeled hUba3 and GST–NEDD8. ³⁵S-Labeled hUba3 and/or ³⁵S-labeled APP–BP1 were cosynthesized for 60 min at 30°C in vitro in a 5 μl reticulocyte lysate transcription/translation system in the presence of 0.35 μg of unlabeled GST–NEDD8, GST–NEDD8(Δ76G), GST–SUMO-1, or GST-Ub, and a part of each (2.5 μl) was analyzed as in Fig. 1A. Arrowheads indicate the ³⁵S-labeled hUba3–GST–NEDD8 complex. (D) Complex formation between ³⁵S-labeled (His)₆–APP–BP1 and ³⁵S-labeled hUba3. ³⁵S-labeled APP–BP1, ³⁵S-labeled (His)₆–APP–BP1, and ³⁵S-labeled hUba3 were synthesized individually for 60 min at 30°C in vitro, and 1 μl of each was analyzed (lanes 1–3), as described in Fig. 1A. After samples (5 μl) of ³⁵S-labeled (His)₆–APP–BP1 or ³⁵S-labeled APP–BP1 had been incubated with ³⁵S-labeled hUba3 (5 μl) for 30 min at 30°C, half of each sample was applied onto a Ni-chelate column. After the column had been washed with 50 mm Na-phosphate buffer (pH 8.0) containing 0.5 m NaCl, the materials eluted with 100 mm EDTA (lanes 4,5) were analyzed as in Fig. 1A.

 Linkage of NEDD8 to Cul-4A in a DTT-insensitive fashion. (A) Alignment of amino acid sequences of two peptides of the rabbit 120-kD protein associated with GST–NEDD8 (protein 5 band in Fig. 1B) and human Cul-4A that we had cloned. Identical amino acids are boxed in black. The Cul-4A cDNA was named Cul-4A(524C), as described in the text. (B) Linkage between ³⁵S-labeled Cul-4A(524C) and GST–NEDD8. ³⁵S-labeled Cul-4A(524C) synthesized in vitro in the presence of unlabeled GST–NEDD8, GST–NEDD8(Δ76G), GST–SUMO-1, or GST–Ub was analyzed as in Fig. 2C. The arrowhead indicates the ³⁵S-labeled Cul-4A(524C)–GST–NEDD8 complex. (C) Linkage between ³⁵S-Labeled Cul-4A(171C) and GST–NEDD8. ³⁵S-labeled Cul-4A(171C) that corresponds to the carboxy-terminal 171 amino acid residues (see Materials and methods) was treated or not with unlabeled GST–NEDD8 and analyzed as in B. The arrowhead indicates the ³⁵S-labeled Cul-4A(171C)–GST–NEDD8 complex.

 Analysis of proteins interacting with NEDD8 in rabbit reticulocyte lysates. (A) Analysis of proteins linked to ³⁵S-labeled NEDD8. After ³⁵S-labeled NEDD8 had been synthesized for 60 min at 30°C in 5 μl of a reticulocyte lysate transcription/ translation system, samples (2.5 μl) of the resultant translational products were subjected directly to SDS-PAGE in the presence (+) or absence (−) of DTT and autoradiographed. (B) SDS-PAGE analysis of proteins purified by GST–NEDD8–GSH–Sepharose 4B chromatography. After reticulocyte lysates had been applied to GSH–Sepharose 4B resin bearing GST or GST–NEDD8, the bound materials were eluted with 40 mm DTT (left) and subsequently with 30 mm GSH (right). Samples were subjected to SDS-PAGE and stained with silver. Proteins bound specifically to GST–NEDD8 are numbered at right (1–5).

 Identification of hUbc12 as a conjugating enzyme for NEDD8. (A) Primary structure of human Ubc12 (Hs) deduced from the nucleotide sequence of a human cDNA and sequence alignment with yeast Ubc12p (Sc) (SWISS-PROT accession no. P52491). The identical amino acids are boxed in black. The amino acid sequences of three peptides of the rabbit 22-kD protein associated with GST–NEDD8 (band 3 in Fig. 1B) are shown above the sequence of hUbc12. The asterisk indicates the essential Cys residue conserved in a family of Ub-conjugating E2 enzymes that becomes linked to Ub in an E2–Ub thioester linkage. (B) Thioester linkage between ³⁵S-labeled hUbc12 and GST–NEDD8. ³⁵S-Labeled hUbc12 synthesized in vitro in the presence of unlabeled GST–NEDD8, GST–NEDD8(Δ76G), GST–SUMO-1, or GST–Ub was analyzed as in Fig. 2. The arrowhead indicates the ³⁵S-labeled hUbc12–GST–NEDD8 complex.