Abstract
Mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they have terminal alpha1-->2-linked N-acetylglucosamine and lack mannose phosphate. In a previous study, Douglas and Ballou (Douglas, R. K., and Ballou, C. E. (1982) Biochemistry 21, 1561-1570) characterized a mutant, mnn2-2, which lacked terminal N-acetylglucosamine in its mannoproteins. The mutant had normal levels of N-acetylglucosaminyltransferase activity, and the partially purified enzyme from wild-type and mutant cells had the same apparent size, heat stability, affinity for substrates, metal requirement, and subcellular location. No qualitative or quantitative differences were found between mutant and wild-type cells in endogenous mannan acceptors and pools of UDP-GlcNAc. Chitin was synthesized at similar rates in wild-type and mutant cells, and the latter did not have a soluble inhibitor of the N-acetylglucosaminyltransferase or a hexosaminidase that could remove N-acetylglucosamine from mannoproteins. Together, the above observations led Douglas and Ballou ((1982) Biochemistry 21, 1561-1570) to postulate that the mutant might have a defect in compartmentation of substrates involved in the biosynthesis of mannoproteins. We determined whether the above mutant phenotype is the result of defective transport of UDP-GlcNAc into Golgi vesicles from K. lactis. Golgi vesicles which were sealed and of the same membrane topographical orientation as in vivo were isolated from wild-type and mnn2-2 mutant cells and incubated with UDP-GlcNAc in an assay in vitro. The initial rate of transport of UDP-GlcNAc into Golgi vesicles from wild-type cells was temperature dependent, saturable with an apparent Km of 5.5 microM and a Vmax of 8.2 pmol/mg of protein/3 min. No transport of UDP-GlcNAc was detected into Golgi vesicles from mutant cells. However, Golgi vesicles from both cells translocated GDP-mannose at comparable velocities, indicating that the above transport defect is specific. In addition to the above defect in mannoproteins, mutant cells were also deficient in the biosynthesis of glucosamine containing lipids.
Highlights
We determined whether the above mutant phenotype is the result of defective transport of UDP-GlcNAc into Golgi vesicles from K. lactis
We determined whether the above mutant phenotype of K. lactis is the result of defective transport of UDP-GlcNAc into Golgi vesicles by comparing the initial rates of transport of UDP-GlcNAc into Golgi vesicles from wild-type and mutant mnn2-2 and mnn2-1 cells
Differential Translocation of UDP-GlcNAc into Golgi Vesicles from Wild-type, mnn2-1, and mnn2-2 Mutant K. lactis—Before transport of nucleotide sugars could be measured into Golgi vesicles from the above cells, it was important to determine that these vesicles were sealed, of the same membrane orientation as in vivo, and of comparable purity
Summary
We determined whether the above mutant phenotype is the result of defective transport of UDP-GlcNAc into Golgi vesicles from K. lactis. Mnn, lacks the terminal N-acetylglucosaminyltransferase activity while the other mutant, mnn, has wild-type levels of this enzyme This latter observation prompted Douglas and Ballou [3] to study the biochemical defect underlying this latter phenotype. Vesicles from all these cells translocated GDP-mannose at a comparable velocity; this indicates that the above defect for UDP-GlcNAc transport is specific In addition to their previously reported lack of N-acetylglucosamine in outer chain of mannoproteins, mnn mutant cells were found deficient in their biosynthesis of glucosamine containing lipids. This phenotype is not secondary to the lack of GlcNAc in glycoproteins since mnn, the glycoprotein GlcNAc-transferase mutant, has the same GlcNAc containing lipids as wild-type.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
