Folding and activity of circularly permuted forms of a polytopic membrane protein

Abstract

The transmembrane subunit of the Glc transporter (IICB Glc ), which mediates uptake and concomitant phosphorylation of glucose, spans the membrane eight times. Variants of IICB Glc with the native N and C termini joined and new N and C termini in the periplasmic and cytoplasmic surface loops were expressed in Escherichia coli. In vivo transport͞in vitro phosphotransferase activities of the circularly permuted variants with the termini in the periplasmic loops 1 to 4 were 35͞58, 32͞37, 0͞3, and 0͞0% of wild type, respectively. The activities of the variants with the termini in the cytoplasmic loops 1 to 3 were 0͞25, 0͞4 and 24͞70, respectively. Fusion of alkaline phosphatase to the periplasmic C termini stabilized membrane integration and increased uptake and͞or phosphorylation activities. These results suggest that internal signal anchor and stop transfer sequences can function as N-terminal signal sequences in a circularly permuted ␣-helical bundle protein and that the orientation of transmembrane segments is determined by the amino acid sequence and not by the sequential appearance during translation. Of the four IICB Glc variants with new termini in periplasmic loops, only the one with the discontinuity in loop 4 is inactive. The sequences of loop 4 and of the adjacent TM7 and TM8 are conserved in all phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system transporters of the glucose family.Escherichia coli ͉ glucose transporter ͉ membrane insertion ͉ phosphotransferase system F olding of multispanning (polytopic) membrane proteins is a sequential process, which according to the current model is coupled to the translation of the protein at the ribosome (1-4). Insertion of membrane proteins requires a special translocation apparatus (translocon), a heterotrimeric complex consisting of the SecY, SecG, and SecE protein subunits in bacteria and the Sec61␣␤␥ subunits in eukaryotes (for a review, see ref. 5). In contrast, folding of globular proteins is a cooperative, posttranslational process whereby chaperones of the DnaK class keep the nascent chains in a folding-competent state until protein synthesis is complete (for a review, see refs. 6 and 7).There are two types of transmembrane segments in polytopic membrane proteins. The signal anchor sequences, which initiate membrane insertion with retention of their N termini in the cytoplasm, and the stop transfer sequences, which terminate the translocation of extracytoplasmic hydrophilic loops and domains. Stop transfer sequences are followed by a high local concentration of positively charged residues, which may arrest further passage of the polypeptide through the translocon (positive inside rule; ref. 8). Transmembrane segments are hydrophobic and assumed to form ␣-helical structures, and they are retained in the translocon, from where they move into the lipid bilayer either sequentially or cooperatively after preassembly in the translocon (reviewed in ref. 9). The first, most N-terminal membrane-spanning sequence can have the orientation of either a...

Folding and Stability of α-Helical Integral Membrane Proteins

“…The generation of other functional polytopic proteins from coexpressed fragments further supports the dispensability of many − but not all topological connections for functional folding. Generating a split variant of a membrane protein can be more complicated: in some instances, the fragments must be coexpressed to achieve wild-type levels of protein in membranes, suggesting that the individual fragments may be unstable or degraded when expressed alone and that lateral interactions between the fragments help to stabilize them in vivo.…”

Membrane Topology of the Multidrug Transporter MdfA: Complementary Gene Fusion Studies Reveal a Nonessential C-Terminal Domain

“…Intuitively, the PhoA moiety might affect the conformation of N11 in such a way that it better recognizes EtBr than chloramphenicol. A related phenomenon has been described recently with circularly permuted variants of the glucose transporter (IICB Glc ), which mediates the uptake and concomitant phosphorylation of glucose (1). The fusion of PhoA to periplasmic segments of IICB Glc increased glucose transport activities.…”

Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS)

“…With the objective to stabilize the interaction between the IIC and IIB domains, circularly permuted [44] and cyclized variants of IICB Glc (C. Siebold, unpublished results) were constructed. In an attempt to break the membrane spanning IIC domain into smaller parts, variants split in the IIC domain were also characterized [34].…”