5'nucleotidase is sorted to the apical domain of hepatocytes via an indirect route.
In hepatocytes, all newly synthesized plasma membrane (PM) proteins so far studied arrive first at the basolateral domain; apically destined proteins are subsequently endocytosed and sorted to the apical domain via transcytosis. A mechanism for the sorting of newly synthesized glycophosphatidylinositol (GPI)-linked proteins has been proposed whereby they associate in lipid microdomains in the trans-Golgi network and then arrive at the apical domain directly. Such a mechanism poses a potential exception to the hepatocyte rule. We have used pulse-chase techniques in conjunction with subcellular fractionation to compare the trafficking of 5' nucleotidase (5NT), an endogenous GPI-anchored protein of hepatocytes, with two transmembrane proteins. Using a one-step fractionation technique to separate a highly enriched fraction of Golgi-derived membranes from ER and PM, we find that both 5NT and the polymeric IgA receptor (pIgAR) traverse the ER and Golgi apparatus with high efficiency. Using a method that resolves PM vesicles derived from the apical and basolateral domains, we find that 5NT first appears at the basolateral domain as early as 30 min of chase. However the subsequent redistribution to the apical domain requires > 3.5 h of chase to reach steady state. This rate of transcytosis is much slower than that observed for dipeptidylpeptidase IV, an apical protein anchored via a single transmembrane domain. We propose that the slow rate of transcytosis is related to the fact that GPI-linked proteins are excluded from clathrin-coated pits/vesicles, and instead must be endocytosed via a slower nonclathrin pathway.
Schell
M J
MJ
Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Maurice
M
M
Stieger
B
B
Hubbard
A L
AL
D015720:5'-Nucleotidase / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D016764:Cell Polarity / Q000502:physiology*; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D016695:Glycosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D016695:Glycosyltransferases; D015720:5'-Nucleotidase