Structurally reduced monosaccharide transporters in an evolutionarily conserved red alga

Abstract

The unicellular red alga Galdieria sulphuraria is a facultative heterotrophic member of the Cyanidiaceae, a group of evolutionary highly conserved extremophilic red algae. Uptake of various sugars and polyols is accomplished by a large number of distinct plasma membrane transporters. We have cloned three transporters [GsSPT1 (G. sulphuraria sugar and polyol transporter 1), GsSPT2 and GsSPT4], followed their transcriptional regulation and assayed their transport capacities in the heterologous yeast system. SPT1 is a conserved type of sugar/H(+) symporter with 12 predicted transmembrane-spanning domains, whereas SPT2 and SPT4 represent monosaccharide transporters, characterized by only nine hydrophobic domains. Surprisingly, all three proteins are functional plasma membrane transporters, as demonstrated by genetic complementation of a sugar uptake-deficient yeast mutant. Substrate specificities were broad and largely redundant, except for glucose, which was only taken up by SPT1. Comparison of SPT1 and truncated SPT1(Delta1-3) indicated that the N-terminus of the protein is not required for sugar transport or substrate recognition. However, its deletion affected substrate affinity as well as maximal transport velocity and released the pH dependency of sugar uptake. In line with these results, uptake by SPT2 and SPT4 was active but not pH-dependent, making a H(+) symport mechanism unlikely for the truncated proteins. We postulate SPT2 and SPT4 as functional plasma membrane transporters in G. sulphuraria. Most likely, they originated from genes encoding active monosaccharide/H(+) symporters with 12 transmembrane-spanning domains.

Figure 1. Northern-blot analysis for GsSPT1, GsSPT2, and GsSPT4

G. sulphuraria was grown autotrophically or heterotrophically on different substrates, as indicated. RNA was isolated during early exponential growth. Ribosomal RNA was used as loading control for total RNA.

Figure 2. Uptake of radiolabelled substrates by yeast cells expressing transporters from G. sulphuraria

GsSPT1 (black), GsSPT2 (dark grey), GsSPT4 (white), and the deletion construct GsSPT1(Δ1–3) (light grey) were expressed in the sugar uptake-deficient yeast strain EBY.VW4000. Transformands were assessed for their uptake capacities for radiolabelled substrates. Uptake of cells expressing the empty vector (below resolution, last column position) as well as of wildtype cells (striped) are given as reference. Assays were performed in triplicate.

Figure 3. Michaelis–Menten (A) and Lineweaver–Burk (B) plots of uptake kinetics for L-fucose

The sugar uptake-deficient yeast strain EBY.VW4000, expressing GsSPT1 (closed circles), GsSPT2 (crosses), GsSPT4 (closed squares) and the deletion construct GsSPT1(Δ1–3) (open circles) respectively, was incubated with L-fucose concentrations ranging from 0 to 0.2 mM. Assays were performed in triplicate. K_m and V_max values are given in the text.

Figure 4. Time-dependent uptake of L-fucose

The sugar uptake-deficient yeast strain EBY.VW4000, expressing GsSPT1 (closed circles), GsSPT2 (closed triangles), GsSPT4 (closed squares) and the deletion construct GsSPT1(Δ1–3) (open circles) respectively, was incubated with saturating L-fucose concentrations (0.1 mM) for up to 6 min. Assays were performed in triplicate.

Figure 5. pH-dependent uptake of L-fucose

The sugar uptake-deficient yeast strain EBY.VW4000, expressing GsSPT1 (closed circles), GsSPT2 (closed triangles), GsSPT4 (closed squares) and the deletion construct GsSPT1(Δ1–3) (open circles) respectively, was incubated with saturating L-fucose concentrations (0.1 mM) at external pH values ranging from pH 4.5 to 8.0. Assays were performed in duplicate.