Identification of residues important for substrate uptake in a glucose transporter from the filamentous fungus Trichoderma reesei.
The glucose transporter is an important player in cell metabolism that mediates the intracellular uptake of glucose. Here, we characterized the glucose transporter Stp1 from the filamentous fungus Trichoderma reesei. The individual substitution of several conserved residues for Ala in Stp1 corresponding to those interacting with D-glucose in the xylose/H(+) symporter XylE inflicted contrasting effects on its ability to support the growth of an hxt-null yeast on glucose. The targeted change of Phe 50, proximal to the substrate-binding site, was also found to exert a profound effect on the activity of Stp1. In contrast with the charged residues, the substitution of Phe 50 with either the hydrophilic residues Asn and Gln or the small residues Gly and Ala significantly enhanced the transport of glucose and its fluorescent analogue, 2-NBDG. On the other hand, a variant with the three substitutions I115F, F199I and P214L displayed remarkably improved activity on glucose and 2-NBDG transport. Further analysis indicated that the combined mutations of Ile 115 and Pro 214, positioned on the lateral surface of the Stp1 N-domain, fully accounted for the enhanced transport activity. These results provide insight into the structural basis for glucose uptake in fungal sugar transporters.
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, No.27 Shanda South Road, Jinan 250100, Shandong, P. R. China.
Weixin
W
Zhang
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, No.27 Shanda South Road, Jinan 250100, Shandong, P. R. China.
Yanli
Y
Cao
Cancer Research Center, School of Medicine, Shandong University, No.44 Wenhuaxi Road, Jinan 250012, Shandong, P. R. China.
Jing
J
Gong
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, No.27 Shanda South Road, Jinan 250100, Shandong, P. R. China.
Xiaoming
X
Bao
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, No.27 Shanda South Road, Jinan 250100, Shandong, P. R. China.
Guanjun
G
Chen
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, No.27 Shanda South Road, Jinan 250100, Shandong, P. R. China.
Weifeng
W
Liu
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D019943:Amino Acid Substitution; D001692:Biological Transport; D017124:Conserved Sequence; D003847:Deoxyglucose / Q000031:analogs & derivatives / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D051246:Glucose Transport Proteins, Facilitative / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014242:Trichoderma / Q000235:genetics* / Q000378:metabolism*
D051246:Glucose Transport Proteins, Facilitative; D011522:Protons; D003847:Deoxyglucose; D009327:4-Chloro-7-nitrobenzofurazan; D005947:Glucose; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose