title: The N-terminal basolateral targeting signal unlikely acts alone in the differential trafficking of membrane transporters in MDCK cells.
abstract: We have shown previously, using confocal imaging and transport assays, that the N-terminus of sodium-dependent vitamin C transporter 2 (SVCT2) can redirect apical SVCT1 to the basolateral membrane. Here, the SVCT model was used to further characterize the basolateral targeting peptide signal. Both the length (31 amino acids) and sequence accuracy of the N-terminus of SVCT2 were found to be important in basolateral targeting activity, suggesting a structural requirement. However, the N-terminal basolateral targeting sequence did not appear to act alone, based on analyses of heterologous chimeras. Although diverse N-terminal basolateral targeting signals from multipass membrane proteins can all redirect apical protein from the same gene family to the basolateral membrane, none of the N-terminal basolateral targeting signals can redirect the transmembrane and C-terminal regions from a different gene family. Instead, the presence of these heterologous N-terminal basolateral targeting signals affected the trafficking of otherwise apical protein, causing their accumulation in a stable tubulin-like non-actin structure. Nontargeting N-terminal sequences had no effect. Similar protein retention was observed previously and in this study when the C-terminus of apical or basolateral protein was mutated. These results suggest that the N- and C-termini interact, directly or indirectly, within each gene family for basolateral targeting. Circular dichroism and two-dimensional nuclear magnetic resonance analyses both found a lack of regular secondary structure in the conserved N-terminus of SVCT2, consistent with the presence of partner(s) in the targeting unit. Our finding, a departure from the prevailing single-peptide motif model, is consistent with the evolution of basolateral transporters from the corresponding apical genes. The interaction among the N-terminus, its partner(s), and the cellular basolateral targeting machinery needs to be further elucidated.
authors.affiliation: Department of Exercise and Nutrition Sciences, University at Buffalo, Buffalo, NY 14214.|Department of Biochemistry, University at Buffalo, Buffalo, NY 14214.
authors.forename: Shiu-Ming
authors.initials: SM
authors.lastname: Kuo
authors.affiliation: Department of Exercise and Nutrition Sciences, University at Buffalo, Buffalo, NY 14214.
authors.forename: Li-Yuan
authors.initials: LY
authors.lastname: Wang
authors.affiliation: Department of Exercise and Nutrition Sciences, University at Buffalo, Buffalo, NY 14214.
authors.forename: Siyuan
authors.initials: S
authors.lastname: Yu
authors.affiliation: Department of Biochemistry, University at Buffalo, Buffalo, NY 14214.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Campbell
authors.affiliation: Department of Biochemistry, University at Buffalo, Buffalo, NY 14214.
authors.forename: Sujith A
authors.initials: SA
authors.lastname: Valiyaparambil
authors.affiliation: Dept. of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267.
authors.forename: Mark
authors.initials: M
authors.lastname: Rance
authors.affiliation: Department of Biochemistry, University at Buffalo, Buffalo, NY 14214.
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Blumenthal
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D017124:Conserved Sequence; D004285:Dogs; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008954:Models, Biological; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D060347:Sodium-Coupled Vitamin C Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D050506:Mutant Chimeric Proteins; D050505:Mutant Proteins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C555162:SLC23A1 protein, human; C555163:SLC23A2 protein, human; D060347:Sodium-Coupled Vitamin C Transporters; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins