Drosophila vigilin, DDP1, localises to the cytoplasm and associates to the rough endoplasmic reticulum

• Published in: Biochimica et biophysica acta Vol. 1809; no. 1; pp. 46 - 55
• Main Authors: Batlle, Marta, Marsellach, Francesc-Xavier, Huertas, Dori, Azorín, Fernando
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 2011
• Subjects: Animals; Animals, Genetically Modified; Blotting, Western; Cells, Cultured; Cytoplasm - metabolism; DDP1; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Drosophila; Drosophila melanogaster - cytology; Drosophila melanogaster - genetics; Drosophila melanogaster - metabolism; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Embryo, Nonmammalian - embryology; Embryo, Nonmammalian - metabolism; Endoplasmic Reticulum, Rough - metabolism; Endoplasmic Reticulum, Rough - ultrastructure; Female; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Heterochromatin; Heterochromatin - genetics; Heterochromatin - metabolism; Histones - metabolism; Immunohistochemistry; KH-domain; Lysine - metabolism; Male; Methylation; Microscopy, Immunoelectron; Mutation; Protein Binding; Ribosomal Proteins - metabolism; RNA; RNA Interference; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Saccharomyces cerevisiae; Schizosaccharomyces - genetics; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe; Schizosaccharomyces pombe Proteins - genetics; Schizosaccharomyces pombe Proteins - metabolism; Vigilin; KH-domain; Heterochromatin; RNA; Vigilin; Drosophila; DDP1
• ISSN: 1874-9399; 0006-3002; 1876-4320
• DOI: 10.1016/j.bbagrm.2010.10.005

Functional characterisation of vigilin, a highly conserved multi-KH-domain protein that binds RNA and ssDNA, remains elusive and, to some extent, controversial. Studies performed in
Saccharomyces cerevisiae and human cells indicate that vigilin localises to the cytoplasm, binds ribosomes, associates to RER and regulates mRNA translation. On the other hand, we and others reported a contribution to heterochromatin-mediated gene silencing (PEV) and chromosome segregation in
S. cerevisiae,
Drosophila and human cells. Whether this contribution is direct remains, however, unclear. Here, we report that
Drosophila vigilin, DDP1, vastly localises to the cytoplasm, being largely excluded from the nucleus. We also show that DDP1 preferentially associates to RER and co-purifies with several ribosomal proteins, suggesting a contribution to mRNA translation. In light of these results, the contribution of DDP1 to PEV was re-examined. Here, we show that a newly generated null
ddp1
Δ mutation is only a weak suppressor of PEV, which is in contrast with our own previous results showing dominant suppression in the presence of a strong hypomorphic
ddp1
15.1
mutation. Similar results were obtained in the fission yeast
Schizosaccharomyces pombe, where vigilin (Vgl1) also associates to RER, having no significant contribution to PEV at centromeres, telomeres and the mating-type
locus. Altogether, these results indicate that cytoplasmic localisation and association to RER, but not contribution to heterochromatin organisation, are evolutionarily conserved features of vigilin, favouring a model by which vigilin acts in the cytoplasm, regulating RNA metabolism, and affects nuclear functions only indirectly.
►Vigilin is a highly conserved multi-KH-domain protein whose functional characterisation remains controversial. ►Vigilin was proposed to regulate RNA metabolism as well as to contribute to heterochromatin function and chromosome segregation. ►Our results show that vigilin localises to the cytoplasm, where it associates to the RER and binds ribosomes. ►Vigilin is largely excluded from the nucleus and its contribution to heterochromatin function is indirect. ►From this we conclude that vigilin acts in the cytoplasm and affects nuclear functions only indirectly, clarifying a confusing topic.