of yeast with a mutation in the KEX2 gene. These mutant cells synthesized the precursor of the a mating factor but could not proteolytically process it to the functional form, and thus were unable to mate with cells of the opposite mating type (see Figure 22-13). The wild-type KEX2 gene encodes an endoprotease that cleaves the a-factor precursor at a site C-terminal to Arg-Arg and Lys-Arg residues. Using the KEX2 gene as a DNA probe, researchers were able to clone a family of mammalian endoproteases, all of which cleave a protein chain on the C-terminal side of an Arg-Arg or Lys-Arg sequence. One, called furin, is found in all mammalian cells; it processes proteins such as albumin that are secreted by the continuous pathway. In contrast, the PC2 and PC3 endoproteases are found only in cells that exhibit regulated secretion; these enzymes are localized to regulated secretory vesicles and proteolytically cleave the precursors of many hormones at specific sites.

Several Pathways Sort Membrane Proteins to the Apical or Basolateral Region of Polarized Cells

The plasma membrane of polarized epithelial cells is divided into two domains, apical and basolateral; tight junctions located between the two domains prevent the movement of plasma-membrane proteins between the domains (see Figure 6-5). Several sorting mechanisms direct newly synthesized membrane proteins to either the apical or basolateral domain of epithelial cells, and any one protein may be sorted by more than one mechanism. Although these sorting mechanisms are understood in general terms, the molecular signals underlying the vesicle-mediated transport of membrane proteins in polarized cells are not yet known. As a result of this sorting and the restriction on protein movement within the plasma membrane due to tight junctions, distinct sets of proteins are found in the apical or basolateral domain. This preferential localization of certain transport proteins is critical to a variety of important physiological functions, such as absorption of nutrients from the intestinal lumen and acidification of the stomach lumen (see Figures 7-27 and 7-28).

Microscopic and cell-fractionation studies indicate that proteins destined for either the apical or the basolateral membranes are initially located together within the membranes of the trans-Golgi network. In some cases, proteins destined for the apical membrane are sorted into their own transport vesicles that bud from the trans-Golgi network and then move to the apical region, whereas proteins destined for the basolateral membrane are sorted into other vesicles that move to the basolateral region. The different vesicle types can be distinguished by their protein constituents, including distinct Rab and v-SNARE proteins, which apparently target them to the appropriate plasma-membrane domain. In this mechanism, segregation of proteins destined for either the apical or basolateral membranes occurs as cargo proteins are incorporated into particular types of vesicles budding from the trans-Golgi network.

Such direct basolateral-apical sorting has been investigated in cultured Madin-Darby canine kidney (MDCK) cells, a line of cultured polarized epithelial cells (see Figure 6-6). In MDCK cells infected with the influenza virus, progeny viruses bud only from the apical membrane, whereas in cells infected with vesicular stomatitis virus (VSV), progeny viruses bud only from the basolateral membrane. This difference occurs because the HA glycoprotein of influenza

► FIGURE 17-26 Sorting of proteins destined for the apical and basolateral plasma membranes of polarized cells.

When cultured MDCK cells are Infected simultaneously with VSV and Influenza virus, the VSV G glycoprotein (purple) Is found only on the basolateral membrane, whereas the influenza HA glycoprotein (green) is found only on the apical membrane. Some cellular proteins (orange circle), especially those with a GPI anchor, are likewise sorted directly to the apical membrane and others to the basolateral membrane (not shown) via specific transport vesicles that bud from the trans-Golgi network. In certain polarized cells, some apical and basolateral proteins are transported together to the basolateral surface; the apical proteins (orange oval) then move selectively, by endocytosis and transcytosis, to the apical membrane. [After K. Simons and A. Wandinger-Ness, 1990, Cell 62:207, and K. Mostov et al., 1992, J. Cell Biol. 116:577.]

VSV G glycoprotein

Trans-Golgi network "

Influenza virus HA glycoprotein

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