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

Journal Article · Wed Nov 01 04:00:00 EST 1989 · Journal of Cell Biology; (USA)

DOI:https://doi.org/10.1083/jcb.109.5.2117· OSTI ID:5102139

Lisanti, M P; Le Bivic, A; Sargiacomo, M; Rodriguez-Boulan, E ^[1]

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1. Cornell Univ. Medical College, NY (USA)

We used domain-selective biotinylation/125I-streptavidin blotting 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.

• OSTI ID: 5102139
• Journal Information: Journal of Cell Biology; (USA), Journal Name: Journal of Cell Biology; (USA) Vol. 109:5; ISSN 0021-9525; ISSN JCLBA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMAL TISSUES
ANIMALS
BETA DECAY RADIOISOTOPES
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL COMPOSITION
CYSTEINE
DAYS LIVING RADIOISOTOPES
DISTRIBUTION
DOGS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
EPITHELIUM
GLYCOPROTEINS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KIDNEYS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
MOLECULAR WEIGHT
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
POST-TRANSLATION MODIFICATION
PROTEINS
RADIOISOTOPES
STEADY-STATE CONDITIONS
THIOLS
TISSUE DISTRIBUTION
TISSUES
TRACER TECHNIQUES
VERTEBRATES