Fusion Proteins with COOH-terminal Ubiquitin Are Stable and Maintain Dual Functionality in Vivo

Abstract

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.

Mimicking proteasomal release of polyglutamine peptides initiates aggregation and toxicity

“…GFP-Ub was enriched in the nucleus but was also present in the cytoplasm and on vesicles (Fig. 1D), similar to the distribution Journal of Cell Science 122 (18) of endogenous Ub (Dantuma et al, 2006;Qian et al, 2002). By contrast, expression of GFP-Ub-Q65 and GFP-Ub-Q112 resulted in the appearance in either the nucleus or cytoplasm of a distinct intracellular structure decorated with fluorescent Ub in a high percentage of the transfected cells.…”

Cutting Edge: Microbial Products Elicit Formation of Dendritic Cell Aggresome-Like Induced Structures in Macrophages

“…We examined DALIS formation in live cells by their expression of a GFP fusion to ubiquitin (11). RAW 264.7 cells were transiently transfected with GFP-ubiquitin and then treated for 8 h with LPS, followed by microscopic analysis.…”

Fusion Proteins for Versatile Antigen Targeting to Cell Surface Receptors Reveal Differential Capacity to Prime Immune Responses

“…Nonremovable (by substitution of Gly 76 ) Ub induces the so-called Ub fusion pathway (18). To act as a degradation signal, Ub must form polyUb chains; although these are generally coupled to Lys 48 , coupling to Lys 29 can be equally or even more important (17,22). Ub coupling, resulting in enhanced degradation (17,22), has been successfully used to enhance CTL responses to Ags encoded by DNA vaccines (23) but, to our knowledge, not so far in recombinant protein vaccines.…”

“…Tandem Ig binding domains of protein G (P2 or P3), which has broad Ig binding capacity across various species and isotypes (16), were inserted to allow for binding of the fusion proteins to Abs. Ub (U) was inserted upstream of the Ags because it can enhance proteasomal degradation of proteins linked to its C terminus (17). Some fusion proteins were also produced with an eGFP (E) to facilitate monitoring of fusion protein binding to and internalization by cells using flow cytometry and microscopy.…”