Fusion proteins with COOH-terminal ubiquitin are stable and maintain dual functionality in vivo.
The ubiquitin (Ub) fusion degradation pathway functions to degrade fusion proteins containing a nonremovable Ub moiety at their NH(2) terminus (Johnson, E. S., Ma, P. C., Ota, I. M., and Varshavsky, A. (1995) J. Biol. Chem. 270, 17442-17456). Here we show that ubiquitin fusion degradation also targets proteins for proteasomal degradation when Ub is present in the middle of fusion proteins (X-Ub-Y), in a process that entails polyubiquitylation of Ub Lys(48). By contrast, fusion proteins bearing COOH-terminal Ub (X-Ub) are metabolically stable. Such fusion proteins, either newly biosynthesized or generated by Ub hydrolases, are reversibly conjugated to heterogeneous target proteins in a manner similar to wild-type Ub. Most importantly, the NH(2)-terminal fusion partner (X) can maintain its structure and function in the formed X-Ub conjugates as inferred from the fluorescence of green fluorescent protein-Ub conjugates and the incorporation of human immunodeficiency virus type 1 Gag-Ub into viral particles. These findings strongly suggest that 26S proteasomes exhibit spatial discrimination of Ub-conjugated proteins, sparing domains extended from the NH(2) terminus of Ub from unfolding and degradation. The multifunctionality of X-Ub fusion proteins opens the possibility for a number of novel practical applications, including the imaging of Ub conjugate formation in living cells.
Qian
Shu-Bing
SB
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0440, USA.
Ott
David E
DE
Schubert
Ulrich
U
Bennink
Jack R
JR
Yewdell
Jonathan W
JW
D000595:Amino Acid Sequence; D000818:Animals; D006367:HeLa Cells; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D006867:Hydrolases; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease