Fractionation and characterization of protein C-terminal prenyl-cysteine methylesterase activities from rabbit brain.

Reversible carboxyl methylation of the C-terminal geranylgeranylcysteine of G25K may regulate its activity and cellular localization. Brain homogenates were examined for enzyme activities which hydrolyze the methyl ester of [3H]methyl-G25K to produce [3H]methanol. Methylesterase activity was detected in both soluble and membrane fractions. The soluble activity was fractionated into at least two distinct activities. One soluble activity appears to be due to the lysosomal protease, cathepsin B, based on sensitivity to certain protease inhibitors, acidic pH optimum, size, and ability to cleave the peptide substrate N alpha-CBZ-Arg-Arg-7-amido-4-methylcoumarin. A second soluble activity, associated with a protein of approximately 25 kDa, exhibits a neutral pH optimum, insensitivity to protease inhibitors, and inhibition by the esterase inhibitor, ebelactone B. The membrane fraction contains larger amounts of a similar methylesterase that may represent the physiologically relevant form of the enzyme.

Design of a membrane protein for site-specific proteolysis: properties of engineered factor Xa protease sites in the lactose permease of Escherichia coli.

Lactose permease is a polytopic membrane transport protein with 12 hydrophobic transmembrane domains connected by hydrophilic loops on the cytoplasmic and periplasmic sides of the membrane. By the use of an active permease mutant devoid of Cys residues (C-less permease), single recognition sites (Ile-Glu-Gly-Arg) for the protease factor Xa (fXa) were engineered into hydrophilic loops 7, 8, and 10 in the C-terminal half of the protein. Mutants carrying single sites inserted at position 255, 259 (loop 7), 283, 286 (loop 8), or 341 (loop 10) exhibit significant lactose accumulation (30-70% of C-less permease) and normal levels of expression in the membrane. However, despite solubilization in dodecyl beta-D-maltoside, none of the mutant permeases is proteolyzed by fXa to a significant extent. Insertion of two recognition sites in tandem at position 255 results in partial cleavage, and remarkably, introduction of three sites in tandem leads to complete proteolysis by fXa. Importantly, mutants with two or three fXa sites at position 255 accumulate lactose to high levels (70% of C-less) and are present in the membrane in amounts comparable to that of C-less permease. The results indicate that hydrophilic loops 7, 8, and 10 are buried in the tertiary structure of the permease where they are inaccessible to protease. Insertion of tandem sites probably facilitates proteolysis by causing loops to become more accessible to the aqueous phase and by increasing the local concentration of protease recognition sites. The approach should be applicable to other polytopic membrane proteins.

Cysteine scanning mutagenesis of putative helix XI in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid in putative transmembrane helix XI was individually replaced with Cys (from Ala347 to Ser366). Fifteen of the 20 mutants are highly functional and accumulate lactose to > 60% of the level achieved by C-less permease, and an additional three mutants, all located at the cytoplasmic end of the helix, exhibit lower but significant lactose accumulation. Cys replacements for Thr348 or Lys358 result in virtually inactive permease. Lys358, however, is not essential for active lactose transport but plays a role in permease folding or membrane insertion by interacting with Asp237. Immunoblots reveal that all mutant proteins are present in the membrane in amounts comparable to C-less with the exception of Lys358-->Cys which is hardly detectable, as expected. The results highlight Thr348 as a potentially important residue for further analysis. Finally, all active mutants were assayed after treatment with the sulfhydryl reagent N-ethyl-maleimide, and results range from nearly complete inhibition to almost 2-fold stimulation. Remarkably, all of the strongly inhibited positions lie on one face of helix XI. The implications of the findings for packing of transmembrane helices in the C-terminal half of the permease are discussed.

Role of the charge pair aspartic acid-237-lysine-358 in the lactose permease of Escherichia coli.

Using a lactose permease mutant devoid of Cys residues (C-less permease), Asp237 and Lys358 were replaced with Cys or other amino acids to pursue the proposal that the two residues form a charge pair [King, S. C., Hansen, C. L., & Wilson, T.H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Individual replacement of Asp237 with Cys, Ala, or Lys or replacement of Lys358 with Cys, Ala, or Asp virtually abolishes active lactose transport. However, simultaneous replacement of both residues with Cys and/or Ala yields permease with high activity. Therefore, neutral amino acid substitutions at either position are detrimental only because they leave the opposing charge unpaired. Strikingly, moreover, when Asp237 is interchanged with Lys358, high activity is observed. The results indicate strongly that Asp237 and Lys358 interact to form a salt bridge and that neither residue nor the salt bridge per se is important for activity. Immunoblots reveal low membrane levels of the active mutants lacking the putative salt bridge, suggesting a role for the salt bridge in either permease folding or stability and raising the possibility that the salt bridge may exist in a folding intermediate but not in the mature protein. Remarkably, however, a mutant with Cys in place of Asp237 is restored to full activity by carboxymethylation which recreates a negative charge at position 237. Pulse-chase analysis and heat-inactivation studies indicate that the stability of the double mutant with Cys at positions 237 and 358 is comparable to C-less. Therefore, the interaction between Asp237 and Lys358 is likely to be important for permease folding and is maintained in the mature protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional interactions between putative intramembrane charged residues in the lactose permease of Escherichia coli.

Using a lactose permease mutant devoid of Cys residue ("C-less permease"), we systematically replaced putative intramembrane charged residues with Cys. Individual replacements for Asp-237, Asp-240, Glu-269, Arg-302, Lys-319, His-322, Glu-325, or Lys-358 abolish active lactose transport. When Asp-237 and Lys-358 are simultaneously replaced with Cys and/or Ala, however, high activity is observed. Therefore, when either Asp-237 or Lys-358 is replaced with a neutral residue, leaving an unpaired charge, the permease is inactivated, but neutral replacement of both residues yields active permease [King, S. C., Hansen, C. L. & Wilson, T. H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Remarkably, moreover, when Asp-237 is interchanged with Lys-358, high activity is observed. The observations provide a strong indication that Asp-237 and Lys-358 interact to form a salt bridge. In addition, the data demonstrate that neither residue nor the salt bridge plays an important role in the transport mechanism. Thirteen additional double mutants were constructed in which a negative and a positively charged residue were replaced with Cys. Only Asp-240-->Cys/Lys-319-->Cys exhibits significant activity, accumulating lactose to 25-30% of the steady state observed with C-less permease. Replacing either Asp-240 or Lys-319 individually with Ala also inactivates the permease, but double mutants with neutral substitutions (Cys and/or Ala) at both positions exhibit essentially the same activity as Asp-240-->Cys/Lys-319-->Cys. In marked contrast to Asp-237 and Lys-358, interchanging Asp-240 and Lys-319 abolishes active lactose transport. The results demonstrate that Asp-240 and Lys-319, like Asp-237 and Lys-358, interact functionally and may form a salt bridge. However, the interaction between Asp-240 and Lys-319 is clearly more complex than the interaction between Asp-237 and Lys-358. In any event, the findings suggest that putative transmembrane helix VII lies next to helices X and XI in the tertiary structure of lactose permease.

Specific disulfide cleavage is required for ubiquitin conjugation and degradation of lysozyme.

Both ubiquitin conjugation and ubiquitin-dependent degradation of chicken egg white lysozyme in a reticulocyte lysate depend on the presence of a reducing agent. We present evidence that the reduction of a specific disulfide bond, namely that at Cys6-Cys127, facilitates ubiquitination and is a prerequisite to the formation of a multiubiquitin chain on one of at least four chain initiation sites on lysozyme. The Cys6-Cys127 disulfide bond in lysozyme can be specifically reduced, and the modified protein can be isolated after carboxymethylation of the 2 resulting cysteines. This modified lysozyme no longer requires the presence of a reducing agent for ubiquitin conjugation and degradation. Inhibition of ubiquitination by the dipeptide Lys-Ala revealed that this modified lysozyme, like the unmodified protein, is recognized via the binding of the ubiquitin protein ligase, E3, to the substrate's N-terminal lysyl residue. Both the rate and the extent of ubiquitin-lysozyme conjugation, however, are significantly higher with this modified substrate. Likewise, ubiquitin-dependent degradation of 6,127-reduced/carboxymethylated lysozyme was 2-4-fold faster than degradation of the unmodified counterpart. These results are consistent with an interpretation that the modified lysozyme mimics an intermediate formed at the rate-limiting step of the degradation of lysozyme in the reticulocyte lysate. Reduction of the Cys6-Cys127 disulfide bond is expected to unhinge the N-terminal region of lysozyme, and we propose that the recognition of this otherwise stable protein by the ubiquitin pathway is due to facilitated binding of E3 that results from such a conformational transition.

Recognition of modified forms of ribonuclease A by the ubiquitin system.

The substrate specificity of the ubiquitin (Ub) conjugation system was explored with regard to recognition of unfolded conformation and/or oxidized methionine residues in six derivatives of bovine RNase A. Based on the following observations, ubiquitination of RNase A substrates by the enzymes in a rabbit reticulocyte extract appears to correlate with unfolded conformation rather than with methionine oxidation. 1) Methionine oxidation in already unfolded forms of RNase A does not enhance ubiquitination. 2) Fluorescence measurements and iodoacetate trapping of free sulfhydryls show that the disulfide bonds of MetSO-RNase A, in which the 4 methionine residues are oxidized to the sulfoxide, are reduced by 2 mM dithiothreitol (DTT) in standard Ub conjugation assays so that this derivative also is unfolded. 3) Although MetSO-RNase A is ubiquitinated in the absence of DTT, its intrinsic fluorescence, cation-exchange properties, and susceptibility to reduction indicate a non-native conformation. 4) Methionine sulfoxide-containing peptides that mimic regions of RNase A fail to inhibit conjugation of 125I-Ub to MetSO-RNase A. Ub adducts to two of the six derivatives (MetSO- and reduced/carboxamidomethylated MetSO-RNase A) increase when DTT is omitted from the reactions. Ubaldehyde, an inhibitor of isopeptidases that disassemble Ub-protein conjugates, increased product yields and reduced or abolished the DTT effect, suggesting that an isopeptidase specific for these two RNase A derivatives may be inactivated by oxidation. Ub conjugates of the other RNase A derivatives also increase with Ub-aldehyde but are unaffected by DTT.