Date of Award
Doctor of Philosophy (PhD)
Nuclear pore complexes are large, uniform organelles with a mass estimated to be on the order of 108 daltons. They are ubiquitous components of the nuclear envelope, providing a channel through which macromolecular transport between the nucleus and cytoplasm occurs. Thus far, almost nothing is known about the proteins that comprise this organelle.
In this thesis, I describe the production of anti-nuclear monoclonal antibodies and their use in identifying some of the constituents of the nuclear pore complex. In particular, I describe studies using an antibody that recognizes predominantly a 62 kD protein (P62) of rat liver nuclei. This protein remains associated with the nuclear pore complex-lamina fraction that results from the treatment of nuclei with DNase, RNase, and nonionic detergent. Immunofluorescence microscopy revealed a strikingly punctate pattern of nuclear rim staining. Using immunogold electron microscopy, p62 was specifically localized to the pore complex. This indicated that pore complexes could be resolved by fluorescence microscopy, and therefore that this assay can be used to screen for antibodies against the pore complex.
Pulse chase analysis of labelled tissue culture cells showed that p62 is synthesized as a soluble cytoplasmic precursor, which is incorporated into the nuclear fraction with an unusually long half time of about six hours. Additionally, p62 is posttranslationally modified by the addition of OlcNAc residues. In fact, it is a member of a family of novel glycoproteins that are labelled in vitro when nuclei are incubated with UDP-3H-galactose in the presence of galactosyl transferase. These proteins have been shown to contain O-linked monosaccharidic OlcNAc residues (Holt, 0.0. and O.W. Han 1986, J. Bioi. Chem. 261:8049-8057). Pulse chase labelling of tissue culture cells with 35S-methionine indicates that most of the sugar is added within 5 min of synthesis when p62 is soluble and cytosolic. Tunicamycin does not prevent glycosylation and the Mr of p62 is not affected by endoglycosidase H. Thus, the addition of GlcNAc appears to be carried out by a cytoplasmically disposed transferase and is clearly distinct from the N- and O-linked glycosylation pathways in the endoplasmic reticulum and Golgi complex.
I have also investigated the subnuclear location of some of the other GlcNAc containing proteins. Using wheat germ agglutinin, as well as another mAb with broad specificity for nuclear GlcNAc-containing proteins, I show by immunofluorescence and protein blotting of subnuclear fractions that there are two sets of GlcNAc containing proteins, distinguished on the basis of subnuclear localization; one consists of about five polypeptides located in the interior of the nucleus, and one probably consists of as many as eight proteins in the pore complex.
Davis, Laura Isabelle, "The Nuclear Pore Complex: Novel Glycoprotein Constituents" (1987). Student Theses and Dissertations. 624.