LDLR-fucosyltransferaseAS fusion protein (312-320)

The Drosophila fat facets gene encodes a deubiquitinating enzyme required during eye development to limit the number of photoreceptors in each facet to eight. Ubiquitin is a small polypeptide that targets proteins for degradation by the proteasome. Deubiquitinating enzymes cleave ubiquitin-protein bonds. In order to investigate the role of FAT FACETS in the ubiquitin pathway, genetic interactions between fat facets and the Drosophila UbcD1 gene were assessed. In addition, three yeast deubiquitinating enzyme genes were tested for their ability to substitute for fat facets in the developing Drosophila eye and for their effects on eye morphology. The results of these experiments support the hypothesis that FAT FACETS activity antagonizes that of the proteolytic machinery. The implications of these results for the specificity of FAF and yeast UBPs are discussed as well.

Bovine herpesvirus-1 infected cell protein 27 (BICP27) was detected predominantly in the nucleolus. The open reading frame of BICP27 was fused with the enhanced yellow fluorescent protein (EYFP) gene to investigate its subcellular localization in live cells and BICP27 was able to direct monomeric, dimeric or trimeric EYFP exclusively to the nucleolus. By constructing a series of deletion mutants, the putative nuclear localization signal (NLS) and nucleolar localization signal (NoLS) were mapped to (81)RRAR(84) and (86)RPRRPRRRPRRR(97) respectively. Specific deletion of the putative NLS, NoLS or both abrogated nuclear localization, nucleolar localization or both respectively. Furthermore, NLS was able to direct trimeric EYFP predominantly to the nucleus but excluded from the nucleolus, whereas NoLS targeted trimeric EYFP primarily to the nucleus, and enriched in the nucleolus with faint staining in the cytoplasm. NLS+NoLS directed trimeric EYFP predominantly to the nucleolus with faint staining in the nucleus. Moreover, deletion of NLS+NoLS abolished the transactivating activity of BICP27 on gC promoter, whereas deletion of either NLS or NoLS did not. The study demonstrated that BICP27 is a nucleolar protein, adding BICP27 to the growing list of transactivators which localize to the nucleolus.

A key step in the signaling mechanism of the mitogen-activated protein kinase/extracellular signal-responsive kinase (ERK) cascade is its translocation into the nucleus where it regulates transcription and other nuclear processes. In an attempt to characterize the subcellular localization of ERK2, we fused it to the 3'-end of the gene expressing green fluorescent protein (GFP), resulting in a GFP-ERK2 protein. The expression of this construct in CHO cells resulted in a nuclear localization of the GFP-ERK2 protein. However, coexpression of the GFP-ERK2 with its upstream activator, MEK1, resulted in a cytosolic retention of the GFP-ERK2, which was the result of its association with MEK1, and was reversed upon stimulation. We then examined the role of the C-terminal region of ERK2 in its subcellular localization. Substitution of residues 312-319 of GFP-ERK2 to alanine residues prevented the cytosolic retention of ERK2 as well as its association with MEK1, without affecting its activity. Most important for the cytosolic retention are three acidic amino acids at positions 316, 319, and 320 of ERK2. Substitution of residues 321-327 to alanines impaired the nuclear translocation of ERK2 upon mitogenic stimulation. Thus, we conclude that residues 312-320 of ERK2 are responsible for its cytosolic retention, and residues 321-327 play a role in the mechanism of ERK2 nuclear translocation.