Isolation and characterisation of smallminded, a Drosophila encoding a new member of the Cdc48p/VCP subfamily of AAA proteins
Introduction
Since the cloning of porcine valosin containing protein (VCP) by Killer and Brownstein (1987), more than 100 genes encoding related proteins have been reported. All of these proteins share either a single or duplicated highly conserved nucleotide binding domain (AAA module), which defines the superfamily, but otherwise show considerable structural heterogeneity beyond these regions (Confalonieri and Duget, 1995). This is reflected in the diversity of their known functions, hence the naming of the family members (Kunau et al., 1993) as ATPases Associated with diverse cellular Activities (AAA). AAA proteins play essential roles in processes as diverse as proteasome function (Dubiel et al., 1992), chaperoning (Arlt et al., 1996), gene regulation (Ohana et al., 1993), organelle biogenesis (Erdmann et al., 1991), membrane fusion (Whiteheart et al., 1994) and cell division (Fröhlich et al., 1991).
Phylogenetic analysis of the AAA family has been used to place its members into six subfamilies of structurally related proteins (AAA server, http://yeamob.pci.chemie,uni-tuebingen.de/kai.html). One such subfamily (Cdc48p/VCP) contains VCP and its homologues from mouse (mVCP; Egerton et al., 1992), Xenopus (P97; Peters et al., 1990) rat (transitional ER-ATPase; Zhang et al., 1994), yeast (Cdc48p; Fröhlich et al., 1991) and the higher plants Arabidopsis (Atcdc48p; Feiler et al., 1995) and soy bean (sVCP; Shi et al., 1995). More distantly related members include the yeast proteins AFG2 (Thorsness et al., 1993), YTA7 (Schnall et al., 1994) and L0919-chroXII (Z73139); archaebacterial proteins SAV, CdcH and mCdc48p (Confalonieri et al., 1994; Bibikov and Oesterhelt, 1994; Bult et al., 1996); Plasmodium CdcATP (M96757) and C. elegans C06A11 and C41C4. (Wilson et al., 1994).
The Cdc48p/VCP subfamily is typical of the AAA superfamily in that its members are characterised primarily by the presence of a duplication of the highly conserved AAA module. Relative to this, the N- and C-terminal regions are often poorly conserved and variable in length. In addition to structural aspects the subfamily also exhibits conserved cellular functions. Some subfamily members are known to be involved in homotypic membrane fusion events. For example, transitional ER-ATPase (TERA) is required for the budding of vesicles from transitional endoplasmic reticulum (Zhang et al., 1994) and p97 plays a role in the reassembly of the post-mitotic Golgi (Acharya et al., 1995; Rabouille et al., 1995). There is also evidence for their involvement in the cell cycle. Mutations in yeast CDC48 lead to a late arrest of the cell cycle (Fröhlich et al., 1991) and this phenotype can be rescued by heterologous expression of the Arabidopsis homologue of cdc48p (Feiler et al., 1995).
Here we present the cloning and characterisation of a Drosophila gene encoding a new member of the Cdc48p/VCP subfamily of AAA proteins.
Section snippets
Isolation of smid mutations
The original mutation was isolated from a screen of GAL4 enhancer-trap lines and the details published elsewhere (Smith and Shepherd, 1996). Seven excision alleles around the site of the P-element insertion e5/e17/e41/e74/e80/e106/e122 were obtained by imprecise excision of the pGAwB element. All mutant stocks were maintained as balanced stocks over either TM3 or TM6b balancer chromosomes. Wild-type data are based on a single outcross of smid alleles to Oregon-R wild-type stock.
Cloning and DNA sequencing
Genomic
Enhancer-trap line c161
As part of a screen of GAL4 enhancer-trap lines designed to identify those in which reporter gene expression is restricted to sensory neurons we uncovered one (C161) which was of particular interest. In addition to a significant expression pattern, this line also had a mutant phenotype associated with the insert. Larvae homozygous for the insertion hatch normally but exhibit poor locomotion and become developmentally arrested as second instar larvae. Homozygous larvae can survive as second
Conclusions
We have described the cloning and sequencing of a new Drosophila gene, smallminded, encoding a member of the AAA superfamily of ATPases. Flies homozygous for mutations of this locus arrest as second instar larvae and have a severely reduced CNS. The predicted amino acid sequence of Smid contains a duplication of the characteristic nucleotide binding domain and is most closely related to members of the Cdc48p/VCP subfamily, suggesting possible roles in the cell cycle and/or homotypic membrane
Unlinked References
Tautz and Pfeifle, 1989
Acknowledgements
The authors would like to thank Douglas Armstrong and Simon Smith for their efforts in the original screen of the Gal4 line. We are also grateful to Mike Bate and Kai Fröhlich for useful discussions. This work was supported by grants from BBSRC, The Wellcome Trust and The Hasselblad Foundation. The nucleotide sequence of the smid cDNA is present in the EMBL database, accession number X99207.
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