Determinants of Translocation and Folding of TreF, a Trehalase of Escherichia coli

Abstract

One isoform of trehalase, TreF, is present in the cytoplasm and a second, TreA, in the periplasm. To study the questions of why one enzyme is exported efficiently and the other is not and whether these proteins can fold in their nonnative cellular compartment, we fused the signal sequence of periplasmic TreA to cytoplasmic TreF. Even though this TreF construct was exported efficiently to the periplasm, it was not active. It was insoluble and degraded by the periplasmic serine protease DegP. To determine why TreF was misfolded in the periplasm, we isolated and characterized Tre ؉ revertants of periplasmic TreF. To further characterize periplasmic TreF, we used a genetic selection to isolate functional TreA-TreF hybrids, which were analyzed with respect to solubility and function. These data suggested that a domain located between residues 255 and 350 of TreF is sufficient to cause folding problems in the periplasm. In contrast to TreF, periplasmic TreA could fold into the active conformation in its nonnative cellular compartment, the cytoplasm, after removal of its signal sequence.Secretory and cytoplasmic proteins differ not only by the signal sequence but also in their folding properties. It is thought that the export competence of secretory proteins is the result of slow folding prior to export. Cytoplasmic proteins are believed to fold more rapidly and thus are not substrates of the cellular secretion apparatus. For a better understanding of the mechanism of translocation, the folding of cytoplasmic and secretory proteins needs to be characterized. Folding of polypeptides is determined by the primary amino acid sequence but also could be influenced by the particular properties of a cellular compartment. However, little is known about whether and how the cytoplasm and the periplasm specifically influence protein folding, i.e. whether there are other elements besides redox state and the presence/absence of ATP. To study these aspects, we used TreA and TreF, the two trehalases of Escherichia coli, as model proteins.Periplasmic TreA is synthesized as a precursor of 565 amino acids. The signal sequence of 30 amino acids is rather long.Mature TreA has a molecular mass of 58 kDa (1, 2). The K m of the purified enzyme is 0.8 mM, the V max is 66 mol of trehalose hydrolyzed/min/mg of protein, and the pH optimum is 5.5 (3). The expression of treA is independent of the presence of trehalose in the growth medium but is stimulated 10-fold at high osmolarity (1, 2). Also, treA is regulated by RpoS, the stationary phase sigma factor (4).Cytoplasmic TreF has 549 amino acids and a molecular mass of 64 kDa. The K m of the purified enzyme is 1.9 mM, the V max is 54 mol of trehalose hydrolyzed/min/mg of protein, and the pH optimum is 6.0. Like treA,

Biochemical and genetic characterization of the pathways for trehalose metabolism in Propionibacterium freudenreichii, and their role in stress response

“…The activity of trehalose glycosyltransferring synthase was tested in both directions, as reported by Qu et al (2004). Assays to investigate the presence of trehalase in cell extracts were performed as described by Uhland et al (2000). The activity of trehalose-6-phosphate phosphorylase and trehalose-6-phosphate hydrolase was assessed by the methods described by Andersson et al (2001) and Rimmele & Boos (1994), respectively.…”

Trehalose-6-phosphate Phosphorylase Is Part of a Novel Metabolic Pathway for Trehalose Utilization in Lactococcus lactis

“…Escherichia coli LE392 was cultivated in Luria-Bertani medium containing 0.2% (w/v) maltose and 10 mM MgSO 4 . The cell cultures were grown according to a Wizard™ Lambda Preps DNA Purification System kit (Promega).…”

Plant-Type Trehalose Synthetic Pathway in Cryptosporidium and Some Other Apicomplexans

“…Most organisms including animals, plants, fungi and bacteria can reutilize trehalose by converting it back to glucose by trehalase [41]–[47]. Among the aveolates, trehalase is present in at least some dinoflagellates and ciliates, but absent in the majority of apicomplexans with a possible exception of Eimeria ( Table 1 ).…”