We are continuing to study the 26S proteasome in S. pombe. In ongoing experiments, we are using antibodies raised against subunit 2 of the 19S regulator to localize the complex in wild-type cells. We also have a strain in which a different 19S subunit has been tagged with HA and used to replace the genomic copy. Immunofluorescence microscopy, using either an HA antibody or antibodies against the 26S complex show identical localization to discrete, punctate structures surrounding the nucleus. We are now using immunoelectron microscopy to further investigate the localization of the 26S complex and to ask whether the staining pattern seen by immunofluorescence corresponds to structures such as ER, or to nuclear pores. For these studies, we have rapidly frozen and freeze-substituted both the wild-type and HA-containing strains and have embedded these well-fixed samples in resins acceptable for immunolabeling (Lowicryl and LR White). Antibodies to HA localize this epitope to the margin of the nucleus, just inside the nuclear envelope. Control cells lack this staining. We hope to use this system to develop reproducible methods to localize many different genes tagged with HA and to elucidate the role that many proteins play in cellular processes.
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