Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network.

Abstract

Abstract. Newly synthesized lysosomal enzymes bind to mannose 6-phosphate receptors (MPRs) in the TGN, and are carded to prelysosomes, where they are released. MPRs then return to the TGN for another round of transport. Rab9 is a ms-like GTPase which facilitates MPR recycling to the TGN in vitro. We show here that a dominant negative form of rab9, rab9 S21N, strongly inhibited MPR recycling in living cells. The block was specific in that the rates of biosynthetic protein transport, fluid phase endocytosis and receptor-mediated endocytosis were unchanged.Expression of rab9 S21N was accompanied by a decrease in the efficiency of lysosomal enzyme sorting. Cells compensated for the presence of the mutant protein by inducing the synthesis of both soluble and membrane-associated lysosomal enzymes, and by internalizing lysosomal enzymes that were secreted by default. These data show that MPRs are limiting in the secretory pathway of cells expressing rab9 S21N and document the importance of MPR recycling and the rab9 GTPase for efficient lysosomal enzyme delivery.M ANNOSE 6-phosphate receptors (MPRs)t deliver newly synthesized, soluble lysosomal enzymes from the TGN to pre-lysosomes CKornfeld and Mellman, 1989;Kornfeld, 1992). Two types of MPRs have been identified to date. One is a 300-kD transmembrane glycoprotein which also binds insulin-like growth factor II; the second is a dimer or tetramer of 45-kD subunits and requires divalent cations for ligand binding in vitro. Both types of MPRs release their ligands upon encountering the low pH within pre-lysosomes, and then return to the Golgi complex to reinitiate another cycle of biosynthetic enzyme transport. MPRs are also present at the cell surface. These receptors are in rapid equilibrium with their intracellular counterparts, and at least the 300-kD MPR is capable of endocytosing extracellular lysosomal hydrolases and delivering them to endocytic compatmtents.Work from a number of laboratories has suggested that the rab family of ras-like GTPases plays a key role in regulating receptor trafficking (Zerial and Stenmark, 1993;Pfeffer, 1992). For example, rab5 has been shown to regulate early endosome fusion both in vitro (Gorvel et al., 1991) and in vivo (Bucci et al., 1992). Rab4 functions in receptor recycling between early endosomes and the cell surface (van der Sluijs et al., 1992). In addition, rabl and its yeast homolog, YPT1, play a key role in the transport of proteins between the ER and the Golgi (Plutner et al., 1991;Tisdale et al., Address all correspondence to S. Pfeffer, Department of Biochemistry, B400, Stanford University Medical School, Stanford, CA 94305-5307.L Abbreviations used in this paper: ECL, enhanced chemiluminescence, man6P, mannose 6-phosphate; MPR, mannose 6-phosphate receptor.1992; Rexach and Schekman, 1991;Segev, 1991). Rab proteins are thought to function in transport vesicle targeting and/or fusion events, because SEC4 mutant yeast strains accumulate secretory vesicles (Novick et al., 1980), anti-FPT/ antibodies inhibit ER-derived tran...

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