The dihydroxyacetone kinase (DhaK) of Escherichia coli consists of three soluble protein subunits. DhaK (YcgT; 39.5 kDa) and DhaL (YcgS; 22.6 kDa) are similar to the N-and C-terminal halves of the ATPdependent DhaK ubiquitous in bacteria, animals and plants. The homodimeric DhaM (YcgC; 51.6 kDa) consists of three domains. The N-terminal dimerization domain has the same fold as the IIA domain (PDB code 1PDO) of the mannose transporter of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The middle domain is similar to HPr and the C-terminus is similar to the N-terminal domain of enzyme I (EI) of the PTS. DhaM is phosphorylated three times by phosphoenolpyruvate in an EI-and HPr-dependent reaction. DhaK and DhaL are not phosphorylated. The IIA domain of DhaM, instead of ATP, is the phosphoryl donor to dihydroxyacetone (Dha). Unlike the carbohydrate-speci®c transporters of the PTS, DhaK, DhaL and DhaM have no transport activity.

The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICB Glc subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICB Glc are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICB Glc and IIA Glc were produced in vivo by RecA intein-mediated trans-splicing. Purified, cyclized IIA Glc and IICB Glc had 100% and 30% of wild-type glucose phosphotransferase activity, respectively. Cyclized IIA Glc displayed increased stability against temperature and GuHCl-induced unfolding. ß

The IICB(Glc) subunit of the glucose transporter acts by a mechanism which couples vectorial translocation with phosphorylation of the substrate. It contains 8 transmembrane segments connected by 4 periplasmic, 2 short, 1 long (80 residues), cytoplasmic loops and an independently folding cytoplasmic domain at the C-terminus. Random DNase I cleavage, EcoRI linker insertion, and screening for transport-active mutants afforded 12 variants with between 46% and 116% of wild-type sugar phosphorylation activity. They carried inserts of up to 29 residues and short deletions in periplasmic loops 1, 2, and 3, in the long cytoplasmic loop 3, and in the linker region between the membrane spanning IIC(Glc) and the cytoplasmic IIB(Glc) domains. Disruption of the gene at the sites of linker insertion decreased the expression level and diminished phosphotransferase activity to between 7% and 32%. IICB(Glc) with a discontinuity in the cytoplasmic loop was purified to homogeneity as a stable complex. It was active only if encoded by a dicistronic operon but not if encoded by two genes on two different replicons, suggesting that spatial proximity of the nascent polypeptide chains is important for folding and membrane assembly.

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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...