Abstract
Employing antisera against various subfractions of rat liver mitochondria (mitoplast, inner membrane, intermembrane, and matrix) as well as metabolically radiolabeled BRL-3A rat liver cells, we undertook a search for the presence of glycoproteins in this major cellular compartment for which little information in regard to glycoconjugates was available. Subsequent to [35S]methionine labeling of BRL-3A cells, a peptide:N-glycosidase-sensitive protein (45 kDa) was observed by SDS-polyacrylamide gel electrophoresis of the inner membrane immunoprecipitate, which was reduced to a molecular mass of 42 kDa by this enzyme. The 45-kDa protein was readily labeled with [2-3H]mannose, and indeed the radioactivity of the inner membrane immunoprecipitate was almost exclusively present in this component. Moreover, antisera directed against mitochondrial NADH-ubiquinone oxidoreductase (complex I) or F1F0-ATPase (complex V) also precipitated a 45-kDa protein from BRL-3A cell lysates as the predominant mannose-radiolabeled constituent. Endo-beta-N-acetylglucosaminidase completely removed the radiolabel from this glycoprotein, and the released oligosaccharides were of the partially trimmed polymannose type (Glc1Man9GlcNAc to Man8GlcNAc). Cycloheximide as well as tunicamycin resulted in total inhibition of radiolabeling of the inner membrane glycoprotein, and moreover, pulse-chase studies employing metrizamide density gradient centrifugation demonstrated that the glycoprotein was initially present in the endoplasmic reticulum (ER) and subsequently appeared in a mitochondrial location. Early movement of the glycoprotein to the mitochondria after synthesis in the ER was also evident from the limited processing undergone by its N-linked oligosaccharides; this stood in contrast to lysosomal glycoproteins in which we noted extensive conversion to complex oligosaccharides. Our findings suggest that the 45-kDa glycoprotein migrates from ER to mitochondria by the previously observed contact sites between the two organelles. Furthermore, the presence of this glycoprotein in at least two major mitochondrial multienzyme complexes would be consistent with a role in mitochondrial translocations.
Highlights
It has been widely recognized for some time that many of the macromolecular components residing in or traversing the ER1-Golgi system on their way to the cell surface or lysosomes are glycoprotein in nature [1], well documented information as to the presence of carbohydrate-containing proteins in other compartments of the cell has only recently emerged
Employing rat liver mitochondrial subfractions as well as the BRL-3A buffalo rat liver cell line, we have by metabolic radiolabeling and immunochemical approaches been able to demonstrate that a major 45-kDa glycoprotein bearing N-linked polymannose carbohydrate is a component of the inner mitochondrial membrane and that it is associated with at least two mitochondrial enzyme complexes
After radiolabeling of the rat liver BRL-3A cell line with [35S]methionine, immunoprecipitates obtained with antisera against mitochondrial subfractions were examined by SDSPAGE with or without prior PNGase digestion to assess the possibility that glycoproteins are present (Fig. 1)
Summary
Many of the macromolecular components residing in or traversing the ER1-Golgi system on their way to the cell surface or lysosomes are glycoprotein in nature [1], well documented information as to the presence of carbohydrate-containing proteins in other compartments of the cell has only recently emerged. Attempts to determine if glycoconjugates are present in mitochondria have been less convincing, and the past literature in this area consists primarily of reports utilizing carbohydrate staining and lectin binding procedures [3,4,5,6,7]. This void in information prompted us in the present study to make an intensive effort to determine if glycoproteins are present in mitochondria and if so to elucidate their localization within the confines of this organelle as well as to define the nature of their carbohydrate units and the site of their formation. Pulse-chase experiments and studies with inhibitors indicated that this glycoprotein is synthesized in the ER and subsequently is translocated to the mitochondria
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