Using ubiquitin to follow the metabolic fate of a protein.

Abstract

We describe a method that can be used to produce equimolar amounts of two or more specific proteins in a cell. In this approach, termed the ubiquitin/protein/reference (UPR) technique, a reference protein and a protein of interest are synthesized as a polyprotein separated by a ubiquitin moiety. This tripartite fusion is cleaved, cotranslationally or nearly so, by ubiquitin-specific processing proteases after the last residue of ubiquitin, producing equimolar amounts of the protein of interest and the reference protein bearing a C-terminal ubiquitin moiety. In applications such as pulse-chase analysis, the UPR technique can compensate for the scatter of immunoprecipitation yields, sample volumes, and other sources of sample-to-sample variation. In particular, this method allows a direct comparison of proteins' metabolic stabilities from the pulse data alone. We used UPR to examine the N-end rule (a relation between the in vivo half-life of a protein and the identity of its N-terminal residue) in L cells, a mouse cell line. The increased accuracy afforded by the UPR technique underscores insufficiency of the current "half-life" terminology, because in vivo degradation of many proteins deyiates from first-order kinetics. We consider this problem and discuss other applications of UPR.

The Final N-Terminal Trimming of a Subaminoterminal Proline-Containing HLA Class I-Restricted Antigenic Peptide in the Cytosol Is Mediated by Two Peptidases

“…Proteolytic activity was monitored by the increased fluorescence resulting from the release of the AMC group (excitation/emission 380/440 nm). A total of 4 nmol peptide RU1 [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] was incubated with 12 l fractions 29, 31, and 32, respectively, for 20 min at 37°C. The digestion was stopped with 2% TFA and analyzed by MS as above.…”

“…We also searched for proteolytic activities in membranes enriched in ER. Although we observed a detectable proteolytic activity against one of the proteasomal products, RU1 [31][32][33][34][35][36][37][38][39][40][41][42] , it could apparently not rescue the loss of CTL recognition of tumor cells resulting from the inhibition of proteasome and TPP II/PSA activities. Our data suggest that the production of RU1 34 -42 /HLA-B51 is a cytosolic process, involving the proteasome to generate the exact C terminus of the antigenic peptide and TPP II and PSA to trim the N-terminal extensions produced by the proteasome.…”

Stability of the Listeria monocytogenes ActA protein in mammalian cells is regulated by the N-end rule pathway

“…Interestingly, the functions of p60 and ActA are required in the cytosol, and both are relatively stable proteins. LLO and the two phospholipases, PI-PLC and PC-PLC, have N-termini (Lys, Tyr, Trp respectively) that are more destabilizing in the mammalian N-end rule than the N-terminus of p60 (Ser) and ActA (Ala) (Gonda et al, 1989;Le  vy et al, 1996). However, the only known functions of LLO and the phospholipases are required in vacuoles, where presumably they would be protected from degradation via the N-end rule pathway.…”

Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila

“…pUAST-3Flag-Ubr3DUBR construct has a deletion of 222-292aa, and pUAST-3Flag-Ubr3DRing construct has a deletion of 1515-2219aa. YFP-based DIAP1 construct is a modified version of the ubiquitin fusion construct described previously by Lévy et al 39 Briefly, YFP was used as a reference protein to link to the N terminus of ubiquitin and the C terminus of ubiquitin was then linked to DIAP1. After cleavage of the tripartite fusion protein at the C terminus of ubiquitin, equimolar amounts of the unmodified DIAP1 and the reference protein will be produced.…”