Analysis of transport and targeting of syndecan-1: effect of cytoplasmic tail deletions.
Madin-Darby canine kidney (MDCK) cells and Chinese hamster ovary (CHO) cells were transfected with wild-type and cytoplasmic deletion mutants of mouse syndecan-1 to study the requirements for transport and polarized expression of this proteoglycan. Expression in MDCK cells revealed that wild-type syndecan-1 is directed to the basolateral surface via a brefeldin A-insensitive route. A deletion of the last 12 amino acids of the syndecan-1 cytoplasmic tail (CT22) was sufficient to result in the appearance of mutant proteoglycans at both the basolateral and apical cell surfaces. Treatment with brefeldin A was able to prevent apical transport of the mutants. We thus propose that the C-terminal part of the cytoplasmic tail is required for steady-state basolateral distribution of syndecan-1. In CHO cells a deletion of the last 25 or 33 amino acids of the 34-residue cytoplasmic domain (CT9 and CT1, respectively) resulted in partial retention of the mutants in the endoplasmic reticulum (ER). A deletion mutant lacking the last 12 amino acids (CT22) was not retained. Interestingly, the unglycosylated core proteins of the CT9 and CT1 mutants showed a significantly lower apparent molecular weight when analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than wild-type syndecan-1. However, when CHO transfectants expressing the CT1 mutant were incubated with brefeldin A, causing fusion of the ER and Golgi, CT1 ran with an almost equally high apparent molecular weight as the wild-type molecule. This would suggest that syndecan-1 undergoes extensive posttranslational modifications or forms an SDS-resistant dimer/complex after transit from the ER.
Miettinen
H M
HM
Centre for Biotechnology, Turku, Finland.
Edwards
S N
SN
Jalkanen
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D016466:CHO Cells; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D003517:Cyclopentanes / Q000494:pharmacology; D003593:Cytoplasm; D004247:DNA; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D053668:Syndecan-1; D053667:Syndecans; D014162:Transfection
D003517:Cyclopentanes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011509:Proteoglycans; C508242:Sdc1 protein, mouse; D053668:Syndecan-1; D053667:Syndecans; D020126:Brefeldin A; D004247:DNA