Reversible topology of a bifunctional transmembrane protein depends upon the charge balance around its transmembrane domain

Abstract

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminal OmpA signal sequence was fused with TEM beta-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the beta-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor. In the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal beta-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the amino-terminal nuclease domain across the membrane, leaving the carboxyl terminal beta-lactamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.

Topology and Functional Domains of the Yeast Pore Membrane Protein Pom152p

“…Adjacent to the TMS, two positively charged amino acid residues are positioned on its Nterminal side, and two negatively charged amino acid residues flank the C-terminal side. This charge distribution flanking the TMS is similar to that observed in other type II membrane proteins and has been used as a tool for predicting membrane protein topology (22)(23)(24).…”

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

“…At approximately 70 amino acid residues downstream of the kinase domain, there is a stretch of 22 hydrophobic amino acids that seems to have the characteristics of a transmembrane domain which is preceded by one residue each of arginine, lysine and histidine. This region is likely to serve as a stop transfer signal for the insertion of this protein into the membrane (Kim et al, 1994). Recently, Pkn2 was reported to contain a similar transmembrane domain (Udo et al, 1995).…”

Myxococcus xanthus, a gram-negative bacterium, contains a transmembrane protein serine/threonine kinase that blocks the secretion of beta-lactamase by phosphorylation.

“…This hydrophobic sequence is flanked by Gln-Arg-Arg-Arg-Glu (total net charge is + 2) at the amino-terminal side and by Ser-GlnArg-Asn-Asp (total net charge is 0) at the carboxy-termihal side within a sequence of 5 residues. This is a typical feature of a transmembrane domain (Gennity and Inouye 1991;Pugsley 1993;Gafvelin and von Heijne 1994;Kim et al 1994) and predicts that Pkn2 is a transmembrane protein having the kinase domain in the cytoplasm and the carboxy-terminal domain downstream of the transmembrane domain outside the cytoplasmic membrane. Within the putative extracellular domain, the signature sequence PKDRLRAHY, known for mitochondrial energy transfer proteins (Klingenberg 1990;Nelson et al 1993), was found from residue 722 to 730 (underlined in Fig.…”

“…The amino acid sequence deduced from the DNA sequence of pkn2 showed the existence of a stretch of 18 hydrophobic amino acid residues (residues 606-623) preceded by positively charged residues in the middle of the regulatory domain, suggesting that this domain functions as an internal signal sequence (Kim et al 1994). To demonstrate that this region can act as an internal signal sequence, fusion proteins, consisting of a fragment of Pkn2 and the E. coli alkaline phosphatase (PhoA), were constructed before and after the hydrophobic stretch as shown in Figure 4.…”