Methanethiol, a gas with the characteristic smell of rotten cabbage, is a product of microbial methionine degradation. In the human body, methanethiol originates primarily from bacteria residing in the lumen of the large intestine. Selenium-binding protein 1 (SELENBP1), a marker protein of mature enterocytes, has recently been identified as a methanethiol oxidase (MTO). It catalyzes the conversion of methanethiol to hydrogen sulfide (H 2 S), hydrogen peroxide (H 2 O 2 ) and formaldehyde. Here, human Caco-2 intestinal epithelial cells were subjected to enterocyte-like differentiation, followed by analysis of SELENBP1 levels and MTO activity. To that end, we established a novel coupled assay to assess MTO activity mimicking the proximity of microbiome and intestinal epithelial cells in vivo . The assay is based on in situ -generation of methanethiol as catalyzed by a bacterial recombinant l -methionine gamma-lyase (MGL), followed by detection of H 2 S and H 2 O 2 . Applying this assay, we verified the loss and impairment of MTO function in SELENBP1 variants (His329Tyr; Gly225Trp) previously identified in individuals with familial extraoral halitosis. MTO activity was strongly enhanced in Caco-2 cells upon enterocyte differentiation, in parallel with increased SELENBP1 levels. This suggests that mature enterocytes located at the tip of colonic crypts are capable of eliminating microbiome-derived methanethiol.

The HUPI gene codes for the monosacharide/H+ cotransr protein of Chiorella kesslni. The gene is functionally expressed in Schizosaccharomyces pombe. This heterologous system has been used to screen for K. mutants of the Chorela symporter. Since S. pombe transformed with HUPI cDNA showed a 1000-fold increase in sensitivity toward the toxic sugar analogue 2-deoxyglucose, we screened for transformants with a decreased 2-deoxyglucose sensitivity.

The cDNAs HUP1 and HUP2 of Chlorella kessleri code for monosaccharide/H ؉ symporters that can be functionally expressed in Schizosaccharomyces pombe. By random mutagenesis three HUP1 mutants with an increased K m value for D-glucose were isolated. The 40-fold increase in K m of the first mutant is due to the amino acid exchange N436I in putative transmembrane helix XI. Two substitutions were found in a second (G97C/ I303N) and third mutant (G120D/F292L), which show a 270-fold and 50-fold increase in K m for D-glucose, respectively. An investigation of the individual mutations revealed that the substitutions I303N and F292L (both in helix VII) cause the K m shifts seen in the corresponding double mutants. These mutations together with those previously found support the hypothesis that helices V, VII, and XI participate in the transmembrane sugar pathway.Whereas The green alga Chlorella kessleri possesses an inducible transport system, capable of accumulative uptake of a variety of monosaccharides using an electrochemical proton gradient as driving force (1-4). Three cDNAs coding for highly homologous Chlorella monosaccharide/H ϩ symporters were cloned by differential screening (5, 6) and named HUP1Ϫ3 (hexose uptake protein). Their identities have been confirmed by heterologous expression in Schizosaccharomyces pombe (6, 7). Furthermore, the HUP1 transporter retains its uptake activity after solubilization from the membrane of transgenic fission yeast, purification to homogeneity, and reconstitution into proteoliposomes (8, 9).The HUP symporters belong to a large family of substrate transporters, called the "major facilitator superfamily" (10). Members of this major facilitator superfamily are thought to consist of 12 ␣-helical transmembrane segments connected by internal and external loops. Support for this topological model comes from alkaline phosphatase fusion protein analysis of the Escherichia coli lactose permease lacY (11) and N-glycosylation scanning mutagenesis studies on the human glucose facilitator GLUT1 (12). However, hard structural data on the nature of the binding sites and translocation pathways of substrates and cosubstrates have not been obtained. Since no three-dimensional structure of a transporter is in sight, one has to be content with indirect evidence, deduced for example from mutagenesis studies.Structure-function analysis of the HUP1 transporter (13, 14) was carried out in a sugar uptake deficient S. pombe strain (15). Several mutants with an increased K m value for D-glucose uptake were found by site-directed mutagenesis (13) and by polymerase chain reaction random mutagenesis with subsequent selection for decreased sensitivity toward the toxic sugar 2-deoxyglucose (14). The affected amino acids cluster in the middle of the transmembrane helices V (Gln-179), VII (Gln-298 and Gln-299), and XI , with the exception of Asp-44 putatively located at the beginning of the first external loop (Fig. 1). The fact that predominantly acidic amino acids and their amides were identified correlates we...

!. IntroductionThe unicellular green alga Chlorella kessleri possesses an inducible hexose transport system [1], capable of accumulative uptake of a variety of monosaccharides and their analogues using a proton gradient for electrogenic secondary active transport [24]. The cDNA coding for a Chlorella monosaccharide/H + co-transporter was cloned by differential screening [5] and named HUP1 (hexose uptake protein 1). Its identity has been confirmed by heterologous expression in Schizosaccharomyces pombe [6] and in Xenopus oocytes [7]. Furthermore, sugar uptake was detectable in an in vitro vesicle system consisting of plasma membranes of transgenic yeast fused with cytochrome-c oxidase containing proteoliposomes [8]. Immunochemical studies on cross-sections of Chlorella cells localized the majority of the HUP1 protein in the plasma membrane as well [9].The HUP1 symporter belongs to a large family of substrate transporters, called major facilitator superfamily (MFS) [10]. The members of this family are thought to consist of 12 putative c~-helical transmembrane segments connected by internal and external loops. This topological model, originally developed solely from hydropathy plots, is in good agreement with data derived from alkaline phosphatase fusion protein analysis of the Escherichia coli lactose permease lacY [11] and N-glycosylation scanning mutagenesis studies on the human glucose facilitator GLUT1 [12]. However, hard structural data of transport proteins are still missing, therefore, informa-*Corresponding author. Fax: (49) (941) Structure-function analysis of the HUP1 transporter was carried out in S. pombe YGS-B25, a sugar uptake-deficient mutant [13]. Several mutants with increased Km value for glucose were found by site-directed mutagenesis [14] and/or PCR random mutagenesis with subsequent selection for decreased sensitivity towards the toxic sugar 2-deoxyglucose [15]. The amino acids affected clustered in the middle of the transmembrane helices V (Q179), VII (Q298 and Q299) and XI (V433 and N436), with the exception of D44, which is located at the beginning of the first external loop (Fig. 1A).The presence of the HUPI protein alone does not cover the broad specificity of monosaccharide transport in Chlorella.Recently, it has been demonstrated that indeed two other monosaccharide/H ÷ symporters are co-induced by glucose [9]. They were designated HUP2 and HUP3 due to their high homologies to the HUP1 transporter (74 and 92%, respectively). Comparison of HUP1 and HUP2, both functionally expressed in S. pombe YGS-B25, showed that the transporters differ significantly concerning their substrate specificity [9]. All the previously identified residues of HUP1 probably involved in the glucose recognition/transport (see above) are also present in HUP2. This raises the question how the different substrate specificities are determined in the two transporters. To answer this, a set of chimeric proteins was constructed and their substrate specificities were characterized. The results clearly point to a particip...

A well-studied transporter of plant cells is the hexose/H+ symporter of the unicellular alga Chlorella kessleri. Its properties, studied in vivo, are briefly summarized. In part, they are atypical and it has been suggested that this porter acts in an asymmetric way. Three genes coding for Chlorella hexose transport activity have been identified (HUP1, HUP2 and HUP3). HUP1 cDNA expressed in a mutant of Schizosaccharomyces pombe not transporting any D-glucose has been studied in detail. Several mutants with changed Km values for substrate were obtained, some by random polymerase chain reaction mutation and selection for decreased sensitivity towards the toxic sugar 2-deoxyglucose, some by site-directed mutagenesis. The amino acids affected clustered in the centre of the putative transmembrane helices V, VII and XI. Large families of hexose transporter genes are found in higher plants (Arabidopsis, Chenopodium, Ricinus). Their functional role is discussed. Finally, the progress made in studying plant transporters in a vesicle system energized by cytochrome c oxidase is summarized.