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Alveolar macrophages have been shown to bind glycoproteins and synthetic glycoconjugates (neogly- The survival of modified glycoproteins, glycoconjugates, and lysosomal glycosidases in mammalian plasma is known to be determined largely by the nature of the exposed, or terminal, sugar residues associated with the carbohydrate chains. Rapid in vivo clearance of glycosylated macromolecules is sugarspecific, displays saturability, and has all the characteristics of a receptor-mediated process. Recent evidence from several laboratories suggests that rapid in vivo clearance of glycoproteins is mediated by at least two distinct, anatomically separate, recognition systems. The innovative work of Ashwell and colleagues (1) has revealed in detail the hepatocyte-dependent receptor-mediated plasma clearance of galactose-terminal (i.e., asialo-) glycoproteins. Based on work from several laboratories (2-10), including our own, evidence for a second pathway has emerged. The latter accomodates many lysosomal glycosidases and glycoproteins having mannose and/or N-acetylglucosamine as their respective terminal sugars. This newly described clearance pathway appears to recognize both mannose and N-acetylglucosamine (GIcNAc) because, as shown by Achord et al. (7), glycoproteins having either of the latter two sugars in the terminal position compete with one another for in vivo clearance. An important clue that the two clearance systems (viz., galactose-specific vs. mannose/GlcNAc-specific) are mutually exclusive and mediated by different liver cell types was the observation that, after clearance of lysosomal glycos- (Bistris), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 2-[bis(2-hydroxyethyl)aminolethanesulfonic acid (Bes), 1,4-piperazinediethanesulfonic acid (Pipes), trypsin, horseradish peroxidase (type VI), ovalbumin and RNases A and B were obtained from Sigma. RNase B was further purified by concanavalin ASepharose chromatography as described by Baynes and Wold (9). Fetuin was obtained from GIBCO, and agalacto-fetuin was prepared chemically by the method of Spiro (12). Agalactoorosomucoid was a generous gift from Gilbert Ashwell; mutant mannans (MNN) were kindly provided by Clinton Ballou. Neoglycoproteins were prepared (kindly donated by Y. C. Lee) by the method of Lee et al. (13) (15). Briefly, the animal was anesthetized with Nembutal (30 mg/kg), the pleural cavity was opened, and the trachea was cannulated. Isotonic saline was allowed to enter the lungs by gravity. The pooled lavage fluid from these separate washings (50 ml) was centrifuged at low speed followed by resuspension of the cells in standard incubation medium (see below). The cells were counted by using a standard hemocytometer and viability was checked by using 0.01% trypan blue. The cells were always used within 1 hr of their isolation, and viability was always >90%. The isolated cells were shown to be >85% macrophages by their histochemical characteristics (acid phosphatase, (1-glucuronidase) and by their ability to take up latex beads.

Abstract. The distribution of a number of membrane proteins on plasmalemmal microdomains (microvilli, coated pits) and in endosomes and lysosomes of the proximal tubule epithelial cell was determined in normal rat kidneys by immunofluorescence and immunoelectron microscopy. Two major brush border proteins, 130 and 94 kD, and 3,-glutamyl transpeptidase were detected on the membranes of the microvilli but were not found on membranes of coated pits. Gp330, the Heymann nephritis antigen, and clathrin were localized in coated pits. The lysosomal membrane glycoprotein, lgpl20 (Lewis, V., S. A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100:1839-1847) was restricted to lysosomes where it co-localized with ~-glucuronidase. Endosomes, identified by preloading with HRP injected 5-15 min before rats were killed, did not contain detectable amounts of any antigen tested. The distribution of the same proteins was also determined in rats given sodium maleate, which is known to slow or reduce protein absorption by the proximal tubule and to cause vacuolation of the endocytic apparatus. After maleate treatment the distribution of microvillar and lysosomal markers was unchanged, but the coated pit markers were redistributed--gp330 was concentrated in newly formed apical vacuoles, and clathrin was diffusely distributed in the apical cytoplasm or on apical coated vesicles. These findings indicate that the membrane composition of microvilli, coated pits, endosomes, and lysosomes is distinctive in the proximal tubule cell; and that gp330, unlike other known coated pit membrane components, is not transferred to endosomes during endocytosis. After maleate treatment, the coated pits lose their clathrin coats, and the corresponding membrane is internalized.

The nature of the cytoplasmic coat present on the apical invaginations of the kidney proximal tubule cell was investigated by immuneoverlay and immunocytochemistry of renal brush borders with anticlathrin antibodies. When kidney cortex was prepared for electron microscopy using methods that enhance visualization of clathrin coats, the apical invaginations at the base of the brush border microvilli were seen to be backed by a nearly continuous coating which resembles but is more extensive than the lattice-like clathrin coats found around brain coated vesicles . When isolated brush border fractions were prepared under conditions that preserve the coats, separated by SDS PAGE, and transferred to nitrocellulose, the presence of clathrin heavy and light chains was detected by immuneoverlay using two different affinitypurified anticlathrin IgGs-one that we prepared, which detects only the clathrin light chains, and the other, prepared by Louvard et al. (Louvard,

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