Subcellular Localization of the Inducible Chlorella HUP1 Monosaccharide-H+ Symporter and Cloning of a Co-Induced Galactose-H+ Symporter

Abstract

l h e unicellular green alga Chlorella kesslerican induce monosaccharide-H+ symport catalyzing the energy-dependent transport of o-glucose (D-GIc) and several other pentoses and hexoses across the plasmalemma. l h e gene coding for the inducible HUPl monosaccharide-H+ symporter has been cloned and the protein has been characterized previously. l h e data presented in this paper demonstrate that the presence of the H U P l gene product alone is not sufficient to cover the broad substrate specificity of monosaccharide transport in induced Chlorella cells. Two other HUPgenes are shown to be co-induced i n Chlorella in response to D-CIC i n the medium. l h e cloning of HUP2 and HUP3 cDNA and genomic sequences is described, both being very homologous to HUPl.Modification of the 5' untranslated sequences of full-length cDNA clones of HUPZ and HUP3 allowed the functional expression of both transporters in Schizosaccharomyces pombe. HUPZ was shown to be a galactose-H+ symporter, whereas the substrate specificity of the HUP3 gene product is very similar to that of the HUPl protein. However, HUP3 does not seem to be induced to high levels in Clc-treated Chlorella cells. Results are also presented proving that the product of the H U P l gene is localized in the plasmalemma of o-Glc-induced Chlorella cells and i s absent i n plasma membranes of noninduced cells. lncubation of thin sections of Chlorella cells with anti-HUP1 antibodies and a fluorescence-labeled, second antibody yielded a ring of fluorescence on the surface of Clc-induced Chlorella cells.~~~~~

Importance of the first external loop for substrate recognition as revealed by chimeric Chlorella monosaccharide/H+ symporters

“…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.…”

“…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).…”

“…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].…”

“…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.…”

“…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).…”