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Plasma membrane glycoproteins recycle to the Golgi complex, but the route followed by these proteins is not known. To elucidate the pathway of transport, the involvement of clathrin-coated vesicles was tested. This was accomplished by comparing the traffic of wild type low density lipoprotein receptor (LDLR) and FH 683, a mutant receptor whose endocytosis from the cell surface in coated vesicles is reduced by 90-95%. Wild type LDLR traveled from the cell surface to the sialyltransferase compartment of the Golgi with a half-time of 2.5 h in K562 human leukemia cells expressing receptor from a transfected cDNA. In contrast, FH 683 LDLR recycled to the Golgi at 33% of the wild type rate, suggesting that wild type LDLR is largely transported to the Golgi by a pathway that involves clathrin-coated vesicles. Moreover, because clathrin-coated vesicles that bud from the plasma membrane are transported to endosomes, surface-to-Golgi transport probably involves an endosomal intermediate. Finally, because there was substantial transport of mutant LDLR to the Golgi even though its endocytosis in coated vesicles was greatly reduced, there may be a second pathway of surface-to-Golgi traffic. Our results suggest that wild type LDLR may move from plasma membrane to Golgi by two routes. Two-thirds of the traffic proceeds via a coated vesicle-mediated pathway while the remainder may follow a clathrin-independent pathway.

Transferrin receptor (TfR) follows complex pathways of transport after endocytosis from the cell surface. Most TfR is transported to endosomes and returns rapidly to the cell surface. In addition, approximately 10% of the internalized receptor recycles through the Golgi complex. To examine the role of microtubules in TfR traffic, K562 cultured human leukemia cells treated with nocodazole to depolymerize microtubules were studied. Nocodazole caused a 50% increase in the level of surface TfR, which was due to a change in receptor dynamics. The endocytosis rate in treated cells was 20% of control, indicating that TfR endocytosis via clathrin-coated vesicles was slowed, whereas the recycling of internalized receptors to the cell surface was unaffected. In contrast, nocodazole had little effect on the transport of TfR from the cell surface to the Golgi complex. Thus, the fragmentation and dispersal of the Golgi complex caused by microtubule depolymerization, which does not interrupt secretory traffic through this organelle, also does not block recycling through the Golgi. The decreased TfR endocytosis via coated vesicles and the increased TfR transport to the Golgi caused by nocodazole suggest that either (i) endocytosis via coated vesicles is not the rate-limiting step in transport to the Golgi or (ii) coated vesicles are not a part of this pathway. Finally, because nocodazole inhibits traffic from endosomes to lysosomes, surface-to-Golgi transport probably does not involve a lysosomal intermediate.

The recycling of plasma membrane glycoproteins to the Golgi complex is well established, but it is not clear which Golgi subcompartments receive this traffic. To date, recycling into the trans-Golgi compartment that contains sialyltransferase and the early Golgi region that contains alpha-mannosidase I has been demonstrated. However, transport into other Golgi compartments has not been reported. In this study we tested the return of cell surface glycoproteins to the Golgi galactosyltransferase compartment using the ldlD mutant of Chinese hamster ovary cells. The cation-independent mannose 6-phosphate/insulin-like growth factor-II receptor recycled through this Golgi region with a half-time of 4 h and was transported to the sialyltransferase compartment as well. Because galactosyltransferase and sialyltransferases are probably located in different trans-Golgi regions in Chinese hamster ovary cells, these results suggest that the two compartments each receive recycling traffic or that recycling glycoproteins enter one region and are then transported to the other. The extent of cell surface protein recycling through the galactosyltransferase compartment was also studied. At least 10 different glycoproteins were transported from the cell surface to this Golgi region. Moreover, our results suggest that recycling glycoproteins make up 12-25% of the flux of cell surface glycoproteins through the Golgi galactosyltransferase compartment; the balance is comprised of newly made glycoproteins.

We have previously shown that transferrin receptor (TfR) recycles from the cell surface through the Golgi complex in K562 human leukemia cells. However, little is known about the transport pathway that carries these receptors to the Golgi complex. To learn more about this transport, we studied the effects of treatments that block specific types of vesicular traffic. K562 cells were cultured in test media and the transport of surface TfR to the Golgi complex was assessed by measuring the entry of asialo-TfR into the sialyltransferase compartment of the Golgi complex. Depletion of cellular potassium, which blocks formation of coated vesicles at the cell surface, stimulated asialo-TfR resialylation by 60% over controls, suggesting that coated vesicle formation is not the rate-limiting step in cell surface-to-Golgi transport. Similarly, culture in sodium-free medium, which blocks transport from endosomes to lysosomes, increased asialo-TfR resialylation by 40%, arguing that lysosomes do not lie on the transport pathway. In contrast, incubation of cells in hypertonic medium, which blocks many vesicular transport steps, inhibited TfR resialylation by 40%, confirming the importance of vesicular traffic in transport of asialo-TfR from the cell surface to the Golgi complex. These results are consistent with two possible pathways for cell surface-to-Golgi transport. Receptor could be transported via an endosomal intermediate, with the rate-limiting step occurring at a post-endosomal site. Alternatively, receptor could be transported directly to the Golgi via a pathway that does not involve endosomes.

The recycling of cellular glycoproteins to the site of Golgi mannosidase I, an enzyme of asparagine-linked oligosaccharide synthesis, was studied in K562 human erythroleukemia cells. Cells were metabolically labeled in the presence of deoxymannojirimycin, a reversible inhibitor of Golgi mannosidase I. This generates glycoproteins with immature oligosaccharides in their normal locations. Transport to the mannosidase I compartment was then assessed by testing for the conversion of oligosaccharides into mature forms during reculture without deoxymannojirimycin. Transferrin receptor (TfR) was acted on by mannosidase I during reculture, suggesting that it returned to the region of the Golgi complex where this enzyme resides. The slow rate of this transport (t1/2 greater than 6 h) implies that it is probably different than TfR movement during transferrin internalization (t1/2 = 10-20 min) and TfR transport to the sialyltransferase compartment in the Golgi complex (t1/2 = 2-3 h) (Snider, M. D., and O. C. Rogers, 1985, J. Cell Biol., 100:826-834). The total cell glycoprotein pool was also transported to the mannosidase I compartment with a half-time of 4 h. Because this transport is 5-10 times faster than the rate of de novo glycoprotein synthesis in these cells, it is likely that most of the glycoprotein traffic through the Golgi complex is composed of recycling molecules.

In eukaryotic cells, secretory proteins and glycoproteins migrate from the rough endoplasmic reticulum, their site of synthesis, through Golgi vesicles before being released from the cell. Cellular and viral integral plasma membrane glycoproteins are co-translationally inserted into the rough endoplasmic reticulum membrane and follow a similar pathway to the cell surface. Previous studies using endoglycosidase H (Endo H) suggested that in rat hepatoma cells the vesicular stomatitis virus (VSV) G protein, albumin and transferrin migrate from the rough endoplasmic reticulum to the Golgi apparatus at different rates. Here we show directly that in human hepatoma HepG2 cells, five secreted proteins mature from the rough endoplasmic reticulum to Golgi vesicles at characteristic rates which differ at least threefold. The results are incompatible with bulk-phase movement of the luminal contents of the endoplasmic reticulum, and suggest that there is a membrane-bound receptor that selectively mediates the transport of secretory proteins from the rough endoplasmic reticulum to the Golgi.

The cation-independent mannose 6-phosphate receptor (MPRCI) functions in the packaging of both newly made and extracellular lysosomal enzymes into lysosomes. The subcellular location of MPRCI reflects these two functions; receptor is found in the Golgi complex, in endosomes, and on the cell surface. To learn about the intracellular pathway followed by surface receptor and to study the relationship between the receptor pools, we examined the entry of the surface MPRCI into Golgi compartments that contain sialyltransferase. Sialic acid was removed from surface-labeled K562 cultured human erythroleukemia cells by neuraminidase treatment. When the cells were returned to culture at 37 degrees C, surface MPRCI was resialylated by the cells with a half-time of 1-2 h. Resialylation was inhibited by reduced temperature, a treatment that allows surface molecules to reach endosomes but blocks further transport. These results indicate that surface MPRCI is transported to the sialyltransferase compartment in the Golgi complex. After culture at 37 degrees C, a small fraction (10-20%) of the resialylated receptor was found on the cell surface. Because a similar fraction of the total receptor pool is found on the cell surface, it is likely that cell surface MPRCI mixes with the cellular pool after resialylation. These data also support the idea that extracellular and newly made lysosomal enzymes are transported to lysosomes through a common compartment.