Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

Abstract

By in vitro translation of mRNA''s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of 2 lysosomal hydrolases, .beta.-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA''s for these 2 proteins were translated in the presnece of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were .apprx. 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membrnaes. In cultured rat liver cells, newly synthesized lysosomal hydrolases showed a dual destination; .apprx. 60% of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. The lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their secretion. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. Modified sugar residues probably serve as sorting out signals which address the hydrolases to their lysosomal destination, with final proteolytic cleavage of hydrolase precursors taking place within the lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathepsin D were detected from their differential sensitivity to digestion with endoglycosidasess H and D. Apparently, the hydrolases exported into the medium follow the normal secretory route and some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.