Abstract
The recently cloned human GLUT9 gene, which maps to chromosome 4p15.3-p16, consists of 12 exons coding for a 540-amino acid protein. Based on a sequence entry (NCBI accession number BC018897) and screening of expressed sequence tags, we have cloned an alternative splice variant of GLUT9 from human kidney cDNA. The RNA of this splice variant consists of 13 exons and codes for a putative protein of 512 amino acids (GLUT9DeltaN). The predicted proteins differ only in their N terminus, suggesting a different subcellular localization and possible physiological role. Screening human tissue RNA by reverse transcription-PCR showed that GLUT9 is expressed mainly in kidney, liver, placenta, and leukocytes, whereas GLUT9DeltaN was detected only in kidney and placenta. The GLUT9 protein localized by immunohistochemistry to human kidney proximal tubules, and subcellular fractionation of human kidney revealed the GLUT9 protein in plasma membranes and high density microsomal membranes. Treatment of kidney membrane proteins with peptide N-glycosidase F showed that GLUT9 and GLUT9DeltaN are expressed in vivo. Localization of GLUT9 and GLUT9DeltaN in three kidney-derived cell lines revealed a plasma membrane distribution for GLUT9 in COS-7 and HEK293 cells, whereas GLUT9DeltaN showed a perinuclear pattern and plasma membrane staining in COS-7 and HEK293 cells, respectively. In polarized Madin-Darby canine kidney cells, GLUT9 trafficked to the basolateral membrane, whereas GLUT9DeltaN localized to the apical membrane. Using heterologous expression of GLUT9 in Xenopus oocytes, GLUT9 appears to be a functional isoform with low affinity for deoxyglucose. Deoxyglucose transport mediated by GLUT9 was not inhibited by cytochalasin B. GLUT9 did not bind cytochalasin B as shown by a cytochalasin B binding assay, indicating a similar behavior of GLUT9 compared with GLUT5.
Highlights
The recently cloned human GLUT9 gene, which maps to chromosome 4p15.3-p16, consists of 12 exons coding for a 540-amino acid protein
Our characterization of GLUT9 shows that GLUT9 is a functional isoform, transporting deoxyglucose with a lower affinity compared with GLUT4
This transport was not inhibitable by cytochalasin B; and using a competitive binding assay, we showed that GLUT9 did not bind cytochalasin B
Summary
Previous studies suggested the presence of additional isoforms [5, 6], and the availability of expressed sequence tag data bases and the sequencing and mapping of the human genome allowed the identification of several new members during the last few years, leading to a rather complex picture of glucose/ hexose transport and whole body sugar homeostasis. Based on sequence characteristics and phylogenetic alignments, three subclasses in the family can be distinguished [7] This suggests a broader range of possible substrates and transport characteristics among these new isoforms that is not restricted to hexoses, as shown by the cloning of the Hϩ/myoinositol transporter isoform or GLUT13 [8]. Transport of hexoses across plasma membranes of mammalian cells is mediated by active as well as passive mechanisms,
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
