α-1,6-Mannosylation of N-linked oligosaccharide present on cell wall proteins is required for their incorporation into the cell wall in the filamentous fungus Neurospora crassa.
The enzyme α-1,6-mannosyltransferase (OCH-1) is required for the synthesis of galactomannans attached to the N-linked oligosaccharides of Neurospora crassa cell wall proteins. The Neurospora crassa och-1 mutant has a tight colonial phenotype and a defective cell wall. A carbohydrate analysis of the och-1 mutant cell wall revealed a 10-fold reduction in the levels of mannose and galactose and a total lack of 1,6-linked mannose residues. Analysis of the integral cell wall protein from wild-type and och-1 mutant cells showed that the mutant cell wall had reduced protein content. The och-1 mutant was found to secrete 18-fold more protein than wild-type cells. Proteomic analysis of the proteins released by the mutant into the growth medium identified seven of the major cell wall proteins. Western blot analysis of ACW-1 and GEL-1 (two glycosylphosphatidylinositol [GPI]-anchored proteins that are covalently integrated into the wild-type cell wall) showed that high levels of these proteins were being released into the medium by the och-1 mutant. High levels of ACW-1 and GEL-1 were also released from the och-1 mutant cell wall by subjecting the wall to boiling in a 1% SDS solution, indicating that these proteins are not being covalently integrated into the mutant cell wall. From these results, we conclude that N-linked mannosylation of cell wall proteins by OCH-1 is required for their efficient covalent incorporation into the cell wall.
Maddi
Abhiram
A
Department of Biological Sciences, SUNY, University at Buffalo, New York 14260, USA.
Free
Stephen J
SJ
D001483:Base Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002473:Cell Wall / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009492:Neurospora crassa / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins; D017261:Glycosylphosphatidylinositols; D009844:Oligosaccharides; D008364:Mannosyltransferases