TRANSMEMBRANE TRANSLOCATION OF SUGAR NUCLEOTIDES

KIM EUGENE CREEK, Purdue University

Abstract

Sugar nucleotides, co-substrates for glycosyltransferases, are synthesized by enzymes located in the cytoplasm or nucleus of rat hepatocytes. However, the glycosyltransferases are intrinsic membrane proteins, located primarily in the Golgi apparatus, where their active sites are generally considered to be directed toward the luminal interiors. Sugar nucleotides must somehow cross membranes in order to react with luminal glycosyltransferases. In this study I investigated possible mechanisms to translocate sugar nucleotides across intracellular membranes from rat liver. Emphasis is on mechanisms of translocation of the sugar nucleotide CMP-sialic acid across membranes of isolated Golgi apparatus. I initially explored the possibility that CMP-sialic acid is made available to sialyltransferases located in the lumens of the Golgi apparatus cisternae by direct synthesis of sugar nucleotide inside this cellular compartment. However, I found that 75% of the cellular CMP-sialic acid synthetase activity was recovered in isolated nuclei and less than 1% was found associated with Golgi apparatus. The synthetase present was extrinsic to the cytoplasmic side of the membranes and therefore any CMP-sialic acid formed would not be directly available to luminal sialyltransferases. The involvement of dolichol or retinol as lipid intermediates to facilitate the translocation of CMP-sialic acid across intracellular membranes was investigated next. Golgi apparatus were unable to catalyze the synthesis of lipid carriers of either the dolichol or retinol type when incubated in the presence of CMP-sialic acid. However, in the presence of GDP-mannose, Golgi apparatus did catalyze the synthesis of mannosyl retinyl phosphate and dolichyl mannosyl phosphate. While a role for retinyl phosphate as a lipid intermediate in the translocation of CMP-sialic acid across membranes was not found, other results showed that retinyl phosphate may affect glycoprotein biosynthesis by altering the rate of dolichyl mannosyl phosphate synthesis and breakdown and the rate of hydrolysis of sugar nucleotides. Isolated Golgi apparatus membranes incorporated sialic acid from CMP-sialic acid into endogenous glycolipid and glycoprotein acceptors in the absence of detergent. Sialyl transfer to endogenous glycoproteins was inhibited 50% by 0.5% Triton X-100 and stimulated 3-fold by 0.05% Triton X-100. Incorporation of sialic acid into endogenous glycolipid acceptors was not stimulated by Triton X-100 and was inhibited at concentrations of Triton X-100 greater than 0.2%. Approximately 60% of the sialic acid incorporated into endogenous acceptors of Golgi apparatus membranes in the absence of detergent was resistant to neuraminidase digestion indicative of a luminal orientation. Therefore CMP-sialic acid was assumed to traverse the Golgi apparatus membranes in the absence of detergent. These results were consistent with passive diffusion or facilitated transport as mechanisms to allow CMP-sialic acid to cross the Golgi apparatus membranes and be used to glycosylate endogenous acceptors. The results of these studies demonstrated that the sugar nucleotide CMP-sialic acid was not synthesized in the lumens of the Golgi apparatus cisternae, was not translocated across these membranes by lipid intermediates, but may cross the membranes either by passive diffusion or by a carrier mediated process.

Degree

Ph.D.

Subject Area

Biology

COinS