THE EFFECTS OF CLATHRIN INACTIVATION ON LOCALIZATION OF KEX2 PROTEASEARE INDEPENDENT OF THE TGN LOCALIZATION SIGNAL IN THE CYTOSOLIC TAIL OF KEX2P

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-termina l cytosolic tail. Mutation of the TLS accelerates transport of Kex2p t o the vacuole by an intracellular (SEC1-independent) pathway. in contr ast, inactivation of the clathrin heavy-chain gene CHC1 results in tra nsport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed b y examining the effects of a temperature-sensitive CHC1 allele on traf ficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TL S mutant forms of Kex2p to behave identically. All forms of Kex2p appe ared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasin g to that of wild-type Kex2p. After inactivation of clathrin heavy cha in, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fu sion to the plasma membrane, indicating that transport to the cell sur face was required for degradation by vacuolar proteolysis. Finally, af ter clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both c hc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localizati on are independent of the Kex2p C-terminal cytosolic tail. Although th ese results neither prove nor rule out a direct interaction between th e Kex2 TLS and a clathrin-dependent structure, they do imply that clat hrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.