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PMID: 2808522 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery.

The Journal of cell biology ·Vol. 109 ·No. 5 ·1989-11-00 ·Pages 2117-27

Lisanti MP, Le Bivic A, Sargiacomo M, Rodriguez-Boulan E

Abstract

We used domain-selective biotinylation/125I-streptavidin blotting (Sargiacomo, M., M. P. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. 1989 J. Membr. Biol. 107:277-286), in combination with lectin precipitation, to analyze the apical and basolateral glycoprotein composition of Madin-Darby canine kidney (MDCK) cells and to explore the role of glycosylation in the targeting of membrane glycoproteins. All six lectins used recognized both apical and basolateral glycoproteins, indicating that none of the sugar moieties detected were characteristic of the particular epithelial cell surface. Pulse-chase experiments coupled with domain-selective glycoprotein recovery were designed to detect the initial appearance of newly synthesized glycoproteins at the apical or basolateral cell surface. After a short pulse with a radioactive precursor, glycoproteins reaching each surface were biotinylated, extracted, and recovered via precipitation with immobilized streptavidin. Several basolateral glycoproteins (including two sulfated proteins) and at least two apical glycoproteins (one of them the major sulfated protein of MDCK cells) appeared at the corresponding surface after 20-40 min of chase, but were not detected in the opposite surface, suggesting that they were sorted intracellularly and vectorially delivered to their target membrane. Several "peripheral" apical proteins were detected at maximal levels on the apical surface immediately after the 15-min pulse, suggesting a very fast intracellular transit. Finally, domain-selective labeling of surface carbohydrates with biotin hydrazide (after periodate oxidation) revealed strikingly different integral and peripheral glycoprotein patterns, resembling the Con A pattern, after labeling with sulfo-N-hydroxy-succinimido-biotin. The approaches described here should be useful in characterizing the steady-state distribution and biogenesis of endogenous cell surface components in a variety of epithelial cell lines.

MeSH Terms
Animals Cell Line Cell Membrane/metabolism Cysteine/metabolism Dogs Electrophoresis, Polyacrylamide Gel Glycoproteins/genetics Glycosylation Kidney Membrane Glycoproteins/biosynthesis,genetics,isolation & purification Molecular Weight Protein Processing, Post-Translational Sulfates/metabolism Sulfur Radioisotopes
Chemicals
Glycoproteins Membrane Glycoproteins Sulfates Sulfur Radioisotopes Cysteine
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Lisanti M P
Department of Cell Biology and Anatomy, Cornell University Medical College, New York 10021.
Le Bivic A
Sargiacomo M
Rodriguez-Boulan E
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.109.5.2117
Published
1989-11-00
Pages
2117-27
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2115858
Subset
IM
Grants
NCRR NIH HHS · 507 RR05396 · United States
NIGMS NIH HHS · GM 34107 · United States
NHLBI NIH HHS · HL 37675 · United States
External Links
PubMed source DOI: 10.1083/jcb.109.5.2117 PMC Full Text View journal details
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