Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes.

Abstract

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity . Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied . The Golgi complex fraction contained most of the gaIactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions . The dolichol-dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex . Although the dolicholindependent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction . Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A, suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed .

TcGPXII, a glutathione-dependent Trypanosoma cruzi peroxidase with substrate specificity restricted to fatty acid and phospholipid hydroperoxides, is localized to the endoplasmic reticulum

“…In eukaryotes the ER is the site where secreted and membrane proteins are synthesized, modified and folded (rough ER) and where fatty acid and phospholipids are synthesized and metabolized (smooth ER). Both types of ER have been isolated from trypanosomatids [38]. For many of these processes an oxidized environment is required, and this is reflected in the relatively low glutathione\glutathione disulphide ratio found in the lumen of the ER (approx.…”

The intracellular pathway and assembly of newly formed variable surface glycoprotein of Trypanosoma brucei.

“…As with other membrane-associated proteins, pre-VSG is synthesized with a rather long 30-40 amino acid hydrophobic NH2-terminal signal peptide, which is subsequently cleaved in the rough ER, where the molecule is synthesized (25). However, unlike eukaryotes, core glycosylation in trypanosomes appears to take place not in the rough ER but rather in some other organelle (tentatively assumed to be the smooth ER) (16). From pulse-chase experiments with L-[35S~methionine (Fig.…”

“…Its incorporation itito VSG is mediated by a dolichol monophosphate intermediate (9) and is inhibited by the antibiotic tunicamycin (11)(12)(13)(14). Recently, using highly enriched Golgi and endoplasmic reticulum (ER) fractions isolated from African trypanosomes (15,16), we tentatively concluded that inhibition of N-acetylglucosaminyl transferase by tunicamycin Al occurred only in the smooth ER subfraction, suggesting that core glycosylation of the VSG may occur in this organelle (16) and not in the rough ER, as previously assumed (10,11,13). As with any centrifugally prepared smooth ER subfraction, it is difficult to ascertain the ratio of ER to other vesiculated fragments of other membranous organelles.…”

“…Golgi and rough ER fractions were isolated essentially as described by Grab et al (15,16) with the exception that an SW-41 rotor was used during the sucrose density fractionation steps. Protein was determined in NaOH-digested membrane fractions according to the method of Whitaker and Granum (24).…”

Cell fractionation of parasitic protozoa: a review

“…The first one was carried out with African trypanosomes (Grab et al 1984). Transmission electron microscopy showed that the fraction consisted predominantly of smooth surface vesicles and flattened cisternae rather than stacked Golgi cisternae.…”