Glucose transport in the extremely thermoacidophilic Sulfolobus solfataricus involves a high-affinity membrane-integrated binding protein

Abstract

The archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 2.5 to 3.5 on carbon sources such as yeast extracts, tryptone, and various sugars. Cells rapidly accumulate glucose. This transport activity involves a membrane-bound glucose-binding protein that interacts with its substrate with very high affinity (Kd of 0. 43 microM) and retains high glucose affinity at very low pH values (as low as pH 0.6). The binding protein was extracted with detergent and purified to homogeneity as a 65-kDa glycoprotein. The gene coding for the binding protein was identified in the S. solfataricus P2 genome by means of the amino-terminal amino acid sequence of the purified protein. Sequence analysis suggests that the protein is anchored to the membrane via an amino-terminal transmembrane segment. Neighboring genes encode two membrane proteins and an ATP-binding subunit that are transcribed in the reverse direction, whereas a homologous gene cluster in Pyrococcus horikoshii OT3 was found to be organized in an operon. These data indicate that S. solfataricus utilizes a binding-protein-dependent ATP-binding cassette transporter for the uptake of glucose.

FIG. 1

pH dependence of glucose uptake by S. solfataricus cells. Uptake assays were performed at 60°C and 1 μM [¹⁴C]glucose at pH 3 (●), pH 4 (□), pH 5 (■), and pH 6 (◊). The rapid decrease in radioactivity at pH 4 after 3 min was probably due to the metabolic degradation of glucose into rapidly diffusible products.

FIG. 2

Glucose uptake by S. solfataricus cells (A) and binding of glucose to purified GBP (B) in the presence of competing substrates. Uptake and binding assays were performed at 60°C and 1 μM [¹⁴C]glucose in the presence of the indicated concentration of mannose (black bars), 2-deoxyglucose (hatched bars), and galactose (white bars). Cells were preincubated for 30 s with nonlabelled sugars in medium (pH 2.5) without carbon source, and uptake was stopped after 10 s. GBP was preincubated for 1 min with nonlabelled sugars in buffer (pH 2), and the binding reaction was stopped after 2 min.

FIG. 3

pH dependence of glucose binding to S. solfataricus membranes (A) and purified GBP (B). Binding assays were performed at 60°C in the presence of 1 μM [¹⁴C]glucose. The buffers used for the different pH values were 250 mM HCl (pH 0.6) (○ [only in panel B]), 50 mM HCl (pH 1.5) (●), 50 mM glycine HCl (pH 2 [□] and 3 [■], and 50 mM citric acid NaOH (pH 4 [▵] and 5 [▴]).

FIG. 4

Purification of the GBP from S. solfataricus membranes. (A and B) Coomassie brilliant blue (A) and glycoprotein (B) staining of SDS-PAGE gels of Triton X-100-solubilized membrane proteins (lane 1), the glycoprotein fraction eluting from the ConA-Sepharose column (lane 2), and the purified binding protein after FPLC Mono Q (lane 3). The positions of the molecular mass standards are indicated. (C) [¹⁴C]glucose staining of a native PAGE gel of purified GBP (lane 1) and solubilized membrane proteins (lane 2).

FIG. 5

Amino acid sequence of the GBP. (A) Sequence of the amino terminus of the purified protein. Also indicated are the nucleotide sequences of the PCR primers used to clone the gene encoding GBP. An asterisk indicates differences in the nucleotide sequence of S. solfataricus P1 and P2. (B) Complete amino acid sequence of the binding protein derived from the nucleotide sequence found in the S. solfataricus genomic bank. The sequenced amino-terminal fragment is boxed, the positions of the putative transmembrane segments are shaded in grey, and putative glycosylation sites are shaded in black.

FIG. 6

Organization of the genomic region around the GBP of S. solfataricus and its homolog, PH1214, of P. horikoshii OT3. Homologous genes are indicated by the same shading pattern. ORF34, ORF35, PH1215, and PH1216 code for putative membrane proteins, and ORF32 encodes an ATP-binding protein belonging to the ABC superfamily.

FIG. 7

Alignment of the ORF32 product of the ABC operon of S. solfataricus with other ATP-binding proteins (PH0203 of P. horikoshii, MsiK of Streptomyces lividans, and AF084104 of Bacillus firmus). The ABC transporter family signature is doubly underlined, and the Walker A (∗) and Walker B (+) motifs of the nucleotide-binding site are indicated. Residues which are conserved in all four proteins are shaded in black, and residues found in only three of the four aligned proteins are shaded in grey.