N-Glycosylation of the alpha subunit does not influence trafficking or functional activity of the human organic solute transporter alpha/beta.
BACKGROUND
The organic solute transporter (OSTalpha-OSTbeta) is a heteromeric transporter that is expressed on the basolateral membrane of epithelium in intestine, kidney, liver, testis and adrenal gland and facilitates efflux of bile acids and other steroid solutes. Both subunits are required for plasma membrane localization of the functional transporter but it is unclear how and where the subunits interact and whether glycosylation is required for functional activity. We sought to examine these questions for the human OSTalpha-OSTbeta transporter using the human hepatoma cell line, HepG2, and COS7 cells transfected with constructs of human OSTalpha-FLAG and OSTbeta-Myc.


RESULTS
Tunicamycin treatment demonstrated that human OSTalpha is glycosylated. In COS7 cells Western blotting identified the unglycosylated form (approximately 31 kD), the core precursor form (approximately 35 kD), and the mature, complex glycoprotein (approximately 40 kD). Immunofluorescence of both cells indicated that, in the presence of OSTbeta, the alpha subunit could still be expressed on the plasma membrane after tunicamycin treatment. Furthermore, the functional uptake of 3H-estrone sulfate was unchanged in the absence of N-glycosylation. Co-immunoprecipitation indicates that the immature form of OSTalpha interact with OSTbeta. However, immunoprecipitation of OSTbeta using an anti-Myc antibody did not co-precipitate the mature, complex glycosylated form of OSTalpha, suggesting that the primary interaction occurs early in the biosynthetic pathway and may be transient.


CONCLUSIONS
In conclusion, human OSTalpha is a glycoprotein that requires interaction with OSTbeta to reach the plasma membrane. However, glycosylation of OSTalpha is not necessary for interaction with the beta subunit or for membrane localization or function of the heteromeric transporter.
Soroka
Carol J
CJ
Department of Medicine, Yale University School of Medicine, New Haven, CT, USA. carol.soroka@yale.edu
Xu
Shuhua
S
Mennone
Albert
A
Lam
Ping
P
Boyer
James L
JL
D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D026901:Membrane Transport Proteins / Q000032:analysis / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D014162:Transfection
D026901:Membrane Transport Proteins; D021122:Protein Subunits; C476575:organic solute transporter alpha, human; C476577:SLC51B protein, human