Abstract
Ribosomal protein S3 (rpS3) is a 243 amino acid component of the 40S ribosomal small subunit. It has multiple roles in translation and extra-ribosomal functions like apoptosis and DNA repair. RpS3 is secreted only in cancer cell lines. Presently, mass spectrometry analysis revealed rpS3 to be glycosylated at the Asn165 residue. A point mutation at this residue decreased secretion of rpS3 in cancer cell lines. Secretion was also inhibited by the endoplasmic reticulum (ER)-Golgi transport inhibitor Brefeldin A and by Tunicamycin, an inhibitor of N-linked glycosylation. N-linked glycosylation of rpS3 was confirmed as necessary for rpS3 secretion into culture media via the ER-Golgi dependent pathway. RpS3 bound to Concanavalin A, a carbohydrate binding lectin protein, while treatment with peptide-N-glycosidase F shifted the secreted rpS3 to a lower molecular weight band. In addition, the N165G mutant of rpS3 displayed reduced secretion compared to the wild-type. An in vitro binding assay detected rpS3 homodimer formation via the N-terminal region (rpS3:1-85) and a middle region (rpS3:95-158). The results indicate that the Asn 165 residue of rpS3 is a critical site for N-linked glycosylation and passage through the ER-Golgi secretion pathway.