No abstract

We reported earlier that the yeast cytochrome c1 presequence (length: 61 amino acids) directs attached proteins to the mitochondrial intermembrane space and that it appears to contain two functional domains: a ‘matrix‐targeting’ domain, and a ‘sorting’ domain. We have now used gene manipulation together with two different in vivo import assays to map these two domains within the cytochrome c1 presequence. The ‘matrix‐targeting’ domain is contained within the N‐terminal 16 residues (or less); by itself, it directs attached proteins to the matrix. The ‘sorting’ domain extends into the C‐terminal 13 residues of the presequence; while it does not mediate intracellular protein transport by itself, it acts together with the preceding ‘matrix‐targeting’ sequence in sorting attached proteins into the intermembrane space. On replacing the authentic ‘matrix‐targeting’ sequence with artificial sequences of different lengths we found that sorting of proteins between the outer membrane and the intermembrane space is not exclusively determined by the length of the N‐terminal ‘matrix‐targeting’ sequence.

Abstract. MDCK cells display fluid-phase transcytosis in both directions across the cell. Transcytosis of cell surface molecules was estimated by electron microscopic analysis of streptavidin-gold-labeled frozen sections of biotinylated cells. Within 3 h, •10% of the surface molecules, biotinylated on the starting membrane domain, were detected on the opposite surface domain irrespective of the direction of transcytosis. This suggests that the transcytosis rates for surface molecules are equal in both directions across the cell as shown previously for fluid-phase markers.A biochemical assay was established to identify transcytosing glycoproteins in MDCKII-RCA r cells, a ricin-resistant mutant of MDCK. Due to a galactosylation defect, surface glycoproteins of these cells can be labeled efficiently with [3H]galactose. Transcytosis of [3H]galactose-labeled glycoproteins to the opposite membrane domain was detected by surface biotinylation. Detergent-solubilized glycoproteins derivatized with biotin were adsorbed onto streptavidin-agarose and separated by SDS-PAGE. A subset of the cell surface glycoproteins was shown to undergo transcytosis. Transport of these glycoproteins across the cell was time and temperature dependent. By comparative twodimensional gel analysis, three classes of glycoproteins were defined. Two groups of glycoproteins were found to be transported unidirectionally by transcytosis, one from the apical to the basolateral surface and another from the basolateral to the apical surface. A third group of glycoproteins which has not been described previously, was found to be transported bidirectionally across the cell.

No abstract

Sorting of newly synthesized proteins destined for the apical plasma membrane takes place in the trans‐Golgi network (TGN) in MDCK cells. This process is most likely receptor mediated and requires components that recycle between both compartments. We have developed an assay to detect apical proteins that recycle through the sialyltransferase‐containing TGN. Cell surface glycoproteins were exogalactosylated apically using a mutant cell line derived from MDCK, MDCKII‐RCAr. The mutant exhibits impaired galactosylation of glycoconjugates and thereby allows maximal incorporation of exogenously added galactose in the presence of galactosyltransferase. Upon reculture at 37 degrees C, a time‐dependent increase of sialylated apical surface glycoproteins was observed by lectin binding as well as by the sialic acid‐specific NaIO4/NaB[3H]4 labeling technique. This indicates that some galactosylated surface molecules had returned to the TGN. Recycling through the TGN was blocked, if exogalactosylated cells were incubated at 20 degrees C. Two‐dimensional gel electrophoresis identified three apical proteins which recycle through the TGN, suggesting that this pathway is selective for a subset of the apical surface proteins.