Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini.

Abstract

An Escherichia coli protease designated Tsp (tail-specific protease) has been purified, and its gene has been cloned and sequenced. Tsp specifically degrades a variant of the N-terminal domain of A repressor in which the five C-terminal residues, which are polar in wild type, have been replaced by nonpolar residues. This substrate specificity in vitro parallels the previously reported selective degradation in vivo of N-terminal-domain variants with nonpolar C-terminal residues. The gene sequence and N-terminal protein sequence of Tsp predict a protein of 660 amino acids. The deduced protein sequence of Tsp shows no significant homology to known protease sequences but does show sequence similarity to the human and bovine interphotoreceptor retinoid-binding proteins, which bind hydrophobic ligands.Within cells, different proteins are degraded with half-lives ranging from minutes to days (1). This heterogeneity implies that intracellular degradation is selective, but the mechanisms of this selectivity are incompletely understood. The C-terminal sequences of some proteins can have dramatic effects on their rates of degradation in Escherichia coli (2, 3). For example, the wild-type N-terminal domain of A repressor (residues 1-102) has the polar C-terminal amino acid sequence RSEYE102 and has a half-life in vivo of >10 hr. The rapidly degraded "#105" variant of this protein has the hydrophobic C-terminal sequence WVAAA102 and has a half-life of 15 min (3). To identify possible cellular component(s) responsible for the degradation of proteins with destabilizing C termini, we have purified an E. coli activity that specifically degrades the #105 variant but not the wild-type N-terminal domain. This activity resides in a single, purified polypeptide (Tsp, for tail-specific protease). We have cloned and sequenced the tsp gene.* Based on sequence comparisons, Tsp shows similarities to the human and bovine interphotoreceptor retinoid-binding proteins (IRBPs) but does not resemble any proteases in the protein sequence data base. MATERIALS AND METHODSAssays for Tsp Activity. Tsp activity in crude lysates and column fractions was assayed by measuring the degradation of 35S-labeled substrates (either the #105 variant or the wild-type N-terminal domain of A repressor) to products soluble in 10% (wt/vol) trichloroacetic acid (3). Reaction mixtures contained _9000 cpm of 35S-labeled substrate, a sample of the E. coli fraction in lysis buffer or column buffer, and 0.02% Nonidet P-40 in a volume of 50 p1. Reaction mixtures were incubated at 370C for 1 or 2 hr and were processed as described (3).In more purified fractions, degradation was assayed by the appearance of substrate digestion products following SDS/ PAGE. Wild-type or #105 protein (2 ,ug), which was purified by the method of Lim and Sauer (4), was mixed with a sample in a 10-1LI reaction volume. Reaction mixtures were incubated at 370C for 3-6 hr and were stopped by boiling for 3 min in Laemmli sample buffer (5). Samples were electrophoresed in SDS/16.5% polyacrylam...

Extra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage‐specific protein over‐expressed in Escherichia coli

“…Our findings [5] Ara C C-terminal tail Increased stability [11] Aldehyde dehydrogenase C-terminal tail Increased stability [12] highlight the contribution of extra C-terminal residues in producing recombinant Pfg27 in a native, folded state which is now suitable for biochemical, biophysical and immunological characterization. Structural elements like the 17 residue C-terminal extension may have widespread application in recombinant protein production.…”

“…polar or nonpolar) can play a role in recognition and subsequent action by cellular proteases [4,5]. In many cases, polar residues at the carboxyl terminal are able to prevent recognition by tail-specific proteases [4][5][6][7]. Together, these studies point to a complex and multifactorial nature of protein folding, and indicate that phenomena like protein stability or its lability are not completely understood yet.…”

“…Several studies have shown that the nature of terminal residues in proteins (i.e. polar or nonpolar) can play a role in recognition and subsequent action by cellular proteases [4,5]. In many cases, polar residues at the carboxyl terminal are able to prevent recognition by tail-specific proteases [4][5][6][7].…”

How does tmRNA move through the ribosome?

“…After puri¢cation, tmRNA was preincubated for 10 min at 37³C under the following ionic conditions: 20 mM HEPES^KOH, pH 7.8 (0³C), 3 mM MgCl 2 , 150 mM NH 4 Cl, 2 mM spermidine, 0.05 mM spermine and 4 mM 2-L-mercaptoethanol. The ¢nal concentration of tmRNA was 1 pmol/Wl.…”

The lone S41 family C-terminal processing protease in Staphylococcus aureus is localized to the cell wall and contributes to virulence

“…As a result, the role of Prc is thought to be in maintaining cell-wall integrity. Prc/Tsp has also been shown to play a role in degrading proteins with non-polar C termini (Silber et al, 1992). Other known targets of Gram-negative CTPs include the outer-membrane protein P13 in Borrelia burgdorferi (Noppa et al, 2001).…”