Hannah Nelson

Cloning and expression of a glycine transporter from mouse brain

We have isolated a cDNA clone from a mouse brain library encoding the glycine transporter (GLYT). Xenopus oocytes injected with a synthetic mRNA accumulated [3H]glycine to levels of up to 80‐fold above control values. The uptake was specific for glycine and dependent on the presence of Na+ and Cl− in the medium. The cDNA sequence predicts a highly hydrophobic protein of 633 amino acids with 12 potential transmembrane helices. The predicted amino acid sequence has 40–45% identity to the GABA, noradrenaline, serotonin and dopamine transporters. This implies that all of these neurotransmitter, transporters may have evolved from a common ancestral gene that diverged into the GABA, glycine and catecholamine subfamilies at nearly the same time.

Cloning of the human brain GABA transporter

A cDNA clone encoding a transporter for the neurotransmitter γ‐aminobutyric acid in human brain was cloned and sequenced. The cDNA contains an open reading frame encoding a hydrophobic protein of 599 amino acids with a calculated molecular weight of 67022 Da. Hydropathy analysis revealed twelve potential transmembrane segments. The human protein is highly homologous to the protein from rat brain. Northern hybridization demonstrated a ubiquitous distribution of the transporter in various parts of the brain.

The progenitor of ATP synthases was closely related to the current vacuolar H+-ATPase

The gene encoding the proteolipid of the vacuolar H+‐ATPase of yeast was cloned and sequenced. The deduced amino acid sequence of the yeast protein is highly homologous to that of the proteolipid from bovine chromaffin granules. In contrast to other membrane proteins the transmembrane segments of the bovine and yeast proteolipids were much more conserved than the hydrophilic parts. The fourth transmembrane segment, which contains the DCCD‐binding site, was conserved 100%. Comparison of vacuolar and eubacterial proteolipids revealed a homology which pointed to a common ancestral gene that underwent gene duplication to form the vacuolar proteolipids. Additional support for this notion came from the amino acid sequences of subunits involved in the catalytic sectors of archaebacterial ATP synthase and plant and yeast vacuolar H+‐ATPases, which reveal extensive sequence homology. Slight, but significant, homology between the archaebacterial and eubacterial ATP synthases was observed. These observations might suggest that the progenitor of ATP synthases was closely related to the present vacuolar H+‐ATPases.

A rat brain cDNA encoding the neurotransmitter transporter with an unusual structure

A rat cDNA clone encoding the novel membrane protein of the neurotransmitter transporters family was cloned and sequenced. The cDNA was identified as a transcript of the gene NTT4 of which a partial genomic clone was previously sequenced. Alignment of the amino acid sequence of NTT4 with other members of the neurotransmitter transport family revealed a marked deviattion from the conserved structure of all other members of the family. The largest extracellular loop with a potential glycosylation site was identified between membrane segments 7 and 8. The protein retains the common glycosylated loop between transmembrane helices 3 and 4 in all members of the family. The transcript of NTT4 was found exclusively in the central nervous system and is more abundant in the cerebellum and the cerebral cortex.

The photosystem I-like P840-reaction center of green S-bacteria is a homodimer.

An operon encoding the P840 reaction center of Chlorobium limicola f.sp.thiosulfatophilum has been cloned and sequenced. It contains two structural genes coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the Photosystem I (PS I) reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS I and for the acceptors A(o), A(1) and FeS-center X are conserved. The second protein is related to the PS I subunit carrying the FeS-centers A and B. Since all our efforts to find a gene for a second, large subunit failed, the P840 reaction center probably is homodimeric.

Short External Loops as Potential Substrate Binding Site of γ-Aminobutyric Acid Transporters

While the γ-aminobutyric acid (GABA) transporter GAT1 exclusively transports GABA, GAT2, −3, and −4 also transport β-alanine. Cross-mutations in the external loops IV, V, and VI among the various GABA transporters were performed by site-directed mutagenesis. The affinity of GABA transport as well as inhibitor sensitivity of the modified transporters was analyzed. Kinetic analysis revealed that a cross-mutation in which loop IV of GAT1 was modified to resemble GAT4 resulted in increased affinity to GABA from Km = 8.7 to 2.0 μM without changing the Vmax. A cross-mutation in loop VI, which swapped the amino acid sequence of GAT2 for GAT1, decreased the affinity to GABA (Km, 35 μM). These results suggest that loops IV and VI contribute to the binding affinity of GABA transporters. A substitution of three amino acids in loop V of GAT1 by the corresponding sequence of GAT3 resulted in β-alanine sensitivity of its GABA uptake activity. These three amino acids in loop V seem to participate in the β-alanine binding domain of GAT3. It is suggested that those three external loops (IV, V, and VI) form a pocket in which the substrate binds to the GABA transporters.

Functional analysis of conserved cysteine residues in the catalytic subunit of the yeast vacuolar H+-ATPase

The A subunit of the yeast vacuolar ATPase contains three highly conserved cysteines: Cys-261, Cys-284, and Cys-538. Cys-261 is located within the nucleotide-binding P-loop. Each of the conserved cysteines, and one nonconserved cysteine, Cys-254, were altered to serine by site-directed mutagenesis, and the effects on growth at pH 7.5 were determined. The Cys-254-->Ser, Cys-261-->Ser and the double mutants all grew at pH 7.5 and contained nitrate- and bafilomycin-sensitive ATPase activity. However, the ATPase activities of the Cys-261-->Ser and the double mutants were insensitive to the sulfhydryl group inhibitor, N-ethylmaleimide, demonstrating that Cys-261 is the site of inhibition by N-ethylmaleimide. Changing either Cys-284 or Cys-538 to serine prevented growth at pH 7.5. Cys-284 and Cys-538 thus appear to be essential cysteine residues which are required either for assembly or catalysis.

Cell Biology and Evolution of Proton Pumps

The vacuolar system of eukaryotic cells contains a large number of organelles that are primarily energized by an H+-ATPase that was named V-ATPase. Several genes encoding subunits of the en