JOURNAL zyxwvu OF CELLULAR PHYSIOLOGY 143:l-12 (19901 zy Endocytosis of Wheat Germ Agglutinin Binding Sites From the Cell Surface Into a Tubular Endosomal Network THOMAS j. RAUB,* MARY 10 KOROLY, AND R. MICHAEL ROBERTS zyxw Drug Delivery Systems Research, The Upjohn Company, Kalamazoo, Michigan zyxw 4900 zyxw I (7J.R.); Department of Biochemistry & Molecular Biology, University of Florida, Cainesville, Florida 326 zyxwvuts I0 fT.].R., M.I.K., R. M.R.) By using fluorescence and electron microscopy, the endocytic pathway encoun- tered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane pro- teins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horserad- ish peroxidase-, or ferritin-conjugated WGA to cells at 4°C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface- labeled cells to 37°C resulted in the endocytosis of WGA into peripheral endo- somes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cister- nae (45-60 nm diam.), which did not interconnect the endosornes. After 30 rnin or more label also became localized in a network of anastornosing tubules (45- 60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incu- bated at 18°C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans-Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post-endosomal,tubular reticulum that appears to be separate from the trans-most Golgi saccule. In another paper (Raub et al., 1990) we show that a heterogeneous subclass of surface glycoproteins on Chi- nese hamster ovary (CHO) cells, are internalized and recycled continuously between the plasma membrane and unknown endocytic compartments. On the basis of cell fractionation studies and the absence of degrada- tion of 1251-wheat germ agglutinin (WGA) that had bound to these high molecular weight acidic glycopro- teins (HMWAG), lysosomes apparently were not in- volved. Similar results have been reported from earlier studies in which HMWAG were labeled specifically with anti-DNP IgG or Fab fragments after intact cells had been haptenized with trinitrobenzenesulfonate (Raub et al., 1986; Raub and Roberts, 1986). Previous work (Horst et al., 1980; Fitzgerald et al., 1981) has suggested that the HMWAG are a major class of membrane proteins that are distributed on the cell surface in discrete domains. In CHO cells, evidence has accumulated to suggest that cell surface glycopro- teins are organized within different domains that can be distinguished by using WGA and concanavalin A (Con A) (Juliano and Li, 1978; Emerson and Juliano, 1982). Both lectins become internalized into vesicles, which then become aggregated within the perinuclear region of the CHO cell (Storrie, 1975; Aubin et al., 1980; Storrie and Edelson, 1977; Schwarz et al., 1982; Fitzgerald et al., 1984). While the pathways of inter- Received June 22, 1988; accepted November 29, 1989. *To whom reprint requesMcorrespondenceshould be addressed. R. Michael Roberts is now at Departments of Biochemistry and Animal Sciences, University of Missouri, Columbia, MO 65211. 0 1990 WILEY-LISS, INC.