InteractiveFly: GeneBrief

smallminded: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - smallminded

Synonyms -

Cytological map position - 66A1--22

Function - ATPase

Keywords - Central nervous system, cell cycle

Symbol - smid

FlyBase ID: FBgn0016983

Genetic map position - 3-

Classification - AAA-protein family signature protein

Cellular location - potentially nuclear, possibly also cytoplasmic



Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Smallminded is a member of a large superfamily of related protein: the ATPases Associated with diverse cellular Activities (AAA). Members share a single or duplicated highly conserved nucleotide binding domain that defines their superfamily, but otherwise show considerable structural heterogeneity beyond these regions. Even the members of the subfamily to which Smallminded belongs appear to have a diversity of functions that makes it difficult to begin to even guess the biological function(s) of Smallminded. Members of the Cdc48p/VPC subfamily, the group to which Smallminded belongs, have been convincingly associated with programmed cell death, protein degradation, membrane fusion and cell division. This essay will concern itself with Drosophila Smallminded and C. elegans MAC-1, structurally and possibly functionally the closest known Smallminded-related protein in another species. Yeast and mammalian Smid-related proteins will be dealt with briefly as well.

Smallminded was isolated in a screen of GAL4 enhancer-trap lines designed to identify those genes in which reporter gene expression is restricted to sensory neurons. In addition to a neural expression pattern, this line also has 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 instar larvae for up to 8 weeks without further development before dying. Over the course of larval development both wild-type and C161 nervous systems increase in size but the rate and extent of the growth of the wild-type CNS is far greater than its C161 equivalent. As early as 36 h after hatching the divergence is such that the wild-type CNS is significantly larger than the equivalently aged CNS from C161 larvae (Long, 1998a). In smid mutants, the majority of preparations show no detectable signs of S-phase cells in the CNS. In those that do show evidence of S-phase cells, the number of NBs undergoing DNA synthesis is significantly less than wild-type, and the number of labelled cells associated with each NB is also reduced. Abnormal levels of cell death by apoptosis occur in smid mutants. Apoptosis is accompanied by heightened expression of reaper and head involution defective. The observation that the CNS of some smid individuals still contains a number of enlarged, non-mitotic NBs five days into larval development suggests that only mutant NBs arrested during mitosis become committed to apoptosis, and that the reduction in S-phases observed in mutant NBs results directly from cell cycle arrest rather than from ectopic cell death (Long, 1998b).

Several yeast and mammalian proteins related to Smid have a demonstrable role in the cell cycle. Disruption of the yeast CDC48 locus produces a phenotype characterized by a late arrest mitosis (Frohlich, 1991). Cdc48p is essential for homotypic membrane fusion of the ER (Latterich, 1995), suggesting that the observed mitotic phenotype is a result of the malfunction of the homotypic membrane fusion machinery essential for the progression of the cell cycle. This idea is reinforced by the observation that the vertebrate homolog of Cdc48p, VCP (or p97), functions in homotypic membrane fusion required for the post-mitotic reassembly of Golgi cisternae (Ribouille, 1995). cdc48 mutants also display a failure to duplicate the spindle pole body, thus implicating Cdc48p in the formation of the spindle apparatus. The differential localization of Cdc48p and VCP during respective yeast and mammalian cell cycles is also indicative of such a role (Madeo, 1998). The subcellular localization of both is regulated in a cell cycle-dependent fashion: both are present on the cytosolic surface of the ER at all times; however Cdc48p also enters the nucleus at the end of G1, when duplication of the spindle pole body occurs, whereas VCP is mobilized to the centrosome throughout mitosis. The correlation of subcellular localization with spindle/centrosome localion, i.e. cytosolic for the centrosome and nuclear membrane-associated for the spindle pole body, indicates conservation of this function but divergence of target localization during evolution. Thus it appears that each of these proteins may perform multiple roles during the cell cycle encompassing organelle enlargement prior to mitosis, spindle body/centrosome duplication, nuclear membrane fusion in yeast, or post-mitotic organelle assembly in higher eukaryotes (Long, 1998b and references).

More closely related to Smallminded than the proteins described above is the C. elegans protein MAC-1. MAC-1 was identified as a protein that binds the C. elegans effector of apoptosis, CED-4. The discovery of physical interactions between CED-3, CED-4 and CED-9 helped elucidate the molecular basis of cell death. Wild-type CED-9 interacts with CED-4 and prevents cell death. CED-9 might function by binding and inactivating CED-4. CED9 is homologous to the mammalian Bcl-2 and Bcl-XL proteins, which both promote survival. CED-4 is homologous to the mammalian protein Apaf-1 (Drosophila homolog: Apaf-1-related-killer), which also promotes cell death. This similarity between CED-4 and Apaf-1 is restricted to their amino-termini, which each contain a caspase recruitment domain similar to that found in the prodomains of the C. elegans caspase CED-3 and mammalian caspases like caspase-9. Thus, homophilic interactions between caspase recruitment domains might allow Apaf-1 to interact with caspase-9, and CED-4 with CED-3. Once bound, Apaf-1 promotes the processing and activation of caspase-9, just as CED-4 promotes the activation of CED-3. Immunoprecipitation studies confirm that MAC-1 interacts with CED-4, and also with Apaf-1 (Wu, 1999 and references). MAC-1 can form a multi-protein complex that also includes CED-3 or CED-9. A MAC-1 transgene under the control of a heat shock promoter prevents some natural cell deaths in C. elegans. Because it is premature to draw conclusions as to the function of MAC-1 in cell death in C. elegans (Wu, 1999), this discussion will new turn to the developmental biology of MAC-1.

Inactivation of mac-1 by RNA-mediated interference, a phenomenon that resembles mutation in its effects, causes animals to arrest as L2 larvae. This arrest is similar to that observed in smallminded mutants, but is not related to the ability of MAC-1 to bind CED-4, since it still occurs in ced-3 or ced-4 null mutants. Although most worms arrest permanently, some eventually complete development, perhaps because the double-stranded RNA that inactivates mac-1 is degraded. These mature animals show additional defects, such as the formation of vacuoles in the intestine, and abnormal vulval and gonadal development. It is not known if these problems reflect a widespread requirement for mac-1 in larval development, or are a side effect of slowed development. However, animals that fail to develop because of starvation have not been seen to exhibit these problems. These results show that the requirement for mac-1 during larval development is unlikely to involve the regulation of programmed cell death, since mutations in ced-3 and ced-4 do not suppress the arrest of mac-1(dsRNAi) animals. Since the screen that identified mac-1 was capable of finding proteins that are essential, or which have pleiotropic effects on development, it should not be surprising that MAC-1 appears to have more than one function, and that its major function might be unrelated to programmed cell death (Wu, 1999). In this respect, several members of the AAA family of ATPases, including mammalian VCP, are known to be involved in diverse cellular functions, including cycle regulation, endocytosis, membrane transport and proteasome regulation (Wu, 1999 and references).

What then is the function of Smallminded? Mutants are defective in larval mitoses, and the expression of Smallminded in cells that undergo late embryonic or larval mitosis suggests a general role in promoting mitosis. Whether Drosophila Smallminded will have other roles in cell biology related to apoptosis, membrane dynamics or protein degradation awaits further exploration into the protein and pathway interactions of the Smallminded protein.


DEVELOPMENTAL BIOLOGY

Probing with the smallminded cDNA indicates that a 3.2 kb transcript is expressed at all key developmental stages: its expression being highest during embryogenesis and at lower but similar levels in larvae, pupae and adults (Long, 1998a).

During the earliest stages of embryogenesis (stages 1-10), SMID mRNA is expressed ubiquitously at high levels. The early ubiquitous expression suggest that the mRNA is maternally supplied. Expression is absent from the amnioserosa during germ band extension. After stage 10, smid expression becomes restricted to two specific tissues. Most obvious is the neurogenic ectoderm, where by stage 12 expression of smid is localized to the NBs that have enlarged and are actively dividing. All NBs express smid at this stage. Expression can be detected in NBs up to stage 15, but throughout this period there is a gradual decline in the number of NBs expressing smid. At stage 13 high expression can be seen in NBs of the thoracic and abdominal NBs. By stage 16 expression can no longer be detected in these NBs, but the cephalic NBs continue to express smid. The other cells expressing high levels of smid are in the gonads, which show expression through to hatching and into larval life. Expression of smid in the non-neurogenic ectoderm decreases markedly post stage 12, coincident with the final divisions of the epidermal cells. Small islands of smid expression can be seen in the epidermis post stage 12 in sensory precursor cells (Long, 1998b).

Larval

The GAL4-induced lacZ expression of line C161 was examined. Line C161 contains a P-element inserted into the smallminded gene. At early larval stages, lacZ is expressed in neuroblasts and in sensory neurons. Neuroblast expression is seen in the cephalic and thoracic neuromeres of the central nervous system. Expression is also seen in imaginal discs. Reporter gene expression is not detected in embryonic stages (Long, 1998a).

In larvae, smid expression is again detected in NBs as they begin their post-embryonic program of cell division. As early as 24 h after hatching, smid expression can be detected in the outer proliferation center (OPC) and in a small number of NBs in the cephalic neuromeres, although none is seen in the thoracic neuromeres. By 96 h after hatching, smid is expressed at high levels throughout the cephalic and thoracic neuromeres and in small segmentally repeated sets in the abdominal neuromeres. smid expression is restricted to the NBs, in a pattern that mirrors exactly the pattern of NB activity in larvae. Smid is also expressed in imaginal discs, in adepithelial cells (the precursors of adult mesoderm), and in segmentally repeated histoblast nests (the precursors of the adult ectodermal derivatives in the abdomen). During late larval stages, high smid expression is detected in small clusters of cells associated with muscles and sensory neurons. These cells are progenitors of the adult mesoderm and are the so-called persistent twist cells (Long, 1998b).


EFFECTS OF MUTATION

The CNS of wild-type and homozygous C161 larvae are comparable in size and indistinguishable. Later in development ( post 24 h), significant differences in the size of the CNS become apparent. Over the next 96 h both wild-type and C161 nervous systems increase in size but the rate and extent of the growth of the wild-type CNS is far greater than its C161 equivalent. As early as 36 h after hatching the difference is such that the wild-type CNS is significantly larger than the equivalently aged CNS from C161 larvae (P<0.001). The wild-type CNS can be seen to be significantly larger than the C161 CNS at all subsequent stages of larval development. It is for this reason that the mutated gene has been named smallminded. This observation supports the hypothesis that mutations of smid disrupt the normal patterns of neurogenesis (Long, 1998a).


EVOLUTIONARY HOMOLOGS
A second AAA superfamily member in Drosophila

A Drosophila homolog of the membrane fusion protein CDC48/p97 has been cloned. The open reading frame of the Drosophila homolog encodes a protein 801 amino acids long (TER94), which shows high similarity to the known CDC48/p97 sequences. The chromosomal position of TER94 is 46 C/D. TER94 is expressed in embryo, in pupae and in imago, but is suppressed in larva. In the imago, the immunoreactivity is exclusively present in the head and in the gonads of both sexes. In the head the most striking staining is observed in the entire neuropil of the mushroom body and in the antennal glomeruli. In addition to TER94, sex-specific forms are also detected in the gonads of the imago: p47 in the ovaries and p98 in the testis. TER94/p47 staining is observed in the nurse cells and often in the oocytes, while TER94/p98 staining is present in the sperm bundles. On the basis of its distribution it is suggested that TER94 functions in the protein transport utilizing endoplasmic reticulum and Golgi derived vesicles (Pinter, 1998).

Smid homologs are involved in cell cycle regulation and vesicular dynamics

An Arabidopsis thaliana CDC48 gene has been identified which, unlike the putative mammalian homolog vasolin-containing protein (VCP), functionally complements Saccharomyces cerevisiae cdc48 mutants. CDC48 is an essential gene in S. cerevisiae and genetic studies suggest a role in spindle pole body separation. Biochemical studies link VCP function to membrane trafficking and signal transduction. The AtCDC48 expression pattern is described in a multicellular eukaryote; the zones of cell division, expansion and differentiation are physically separated in higher plants, thus allowing the analysis of in situ expression patterns with respect to the state of cell proliferation. AtCDC48 is highly expressed in the proliferating cells of the vegetative shoot, root, floral inflorescence and flowers, and in rapidly growing cells. AtCDC48 mRNA and the encoded protein are up-regulated in the developing microspores and ovules. AtCDC48 expression is down-regulated in most differentiated cell types. AtCDC48p is primarily localized to the nucleus and, during cytokinesis, to the phragmoplast, a site where membrane vesicles are targeted in the deposition of new cell wall materials. This study shows that the essential cell division function of CDC48 has been conserved by, at least, some multicellular eukaryotes and suggests that in higher plants, CDC48 functions in cell division and growth processes (Feiler, 1995).

Yeast mutants of cell cycle gene cdc48-1 arrest as large budded cells with microtubules spreading aberrantly throughout the cytoplasm from a single spindle plaque. The gene was cloned and disruption proved it to be essential. The CDC48 sequence encodes a protein of 92 kD that has an internal duplication of 200 amino acids and includes a nucleotide binding consensus sequence. Vertebrate VCP has a 70% identity over the entire length of the protein. Yeast Sec18p and mammalian N-ethylmaleimide-sensitive fusion protein, which are involved in intracellular transport, yeast Pas1p, which is essential for peroxisome assembly, and mammalian TBP-1, which influences HIV gene expression, are 40% all identical to each outer in the duplicated region. Antibodies against CDC48 recognize a yeast protein of apparently 115 kD and a mammalian protein of 100 kD. Both proteins are bound loosely to components of the microsomal fraction as described for Sec18p and N-ethylmaleimide-sensitive fusion protein. This similarity suggests that CDC48p participates in a cell cycle function related to that of N-ethylmaleimide-sensitive fusion protein/Sec18p in Golgi transport (Frohlich, 1991).

The fusion of endoplasmic reticulum (ER) membranes in yeast is an essential process required for normal progression of the nuclear cell cycle (karyogamy) and the maintenance of an intact organellar compartment. This process requires a novel fusion machinery distinct from the classic membrane docking/fusion machinery containing Sec17p (alpha-SNAP) and Sec18p (NSF). Cdc48p, a cell-cycle protein with homology to Sec18p, is required in ER fusion. A temperature-sensitive cdc48 mutant is conditionally defective in ER fusion in vitro. Addition of purified Cdc48p restores the fusion of isolated cdc48 mutant ER membranes. It is proposed that Cdc48p is part of an evolutionarily conserved fusion/docking machinery involved in multiple homotypic fusion events (Latterich, 1995).

Golgi cisternae regrow in a cell-free system from mitotic Golgi fragments incubated with buffer alone. Pretreatment with NEM or salt washing inhibits regrowth, but this can be restored either by p97, an NSF-like ATPase, or by NSF together with SNAPs and p115, a vesicle docking protein. The morphology of cisternae regrown with p97 and NSF-SNAPs-p115 differs, suggesting that they play distinct roles in rebuilding Golgi cisternae after mitosis (Rabouille, 1995).

Cdc48p is essential for homotypic endoplasmic reticular fusion in Saccharomyces cerevisiae. It is localized at the endoplasmic reticulum during most of the cell division cycle but concentrates in the nucleus at the G1/S-transition. Its mammalian homolog VCP (valosin-containing protein) alternates between the endoplasmic reticulum and the centrosome in in a cell cycle dependent manner. Though Cdc48p and porcine VCP show a high sequence conservation -- almost 70% of their amino acid residues are identical -- the VCP gene fails to complement a disruption of CDC48. Complementation studies with CDC48 and VCP gene hybrids show that an exchange of the central Cdc48p domain for the central VCP domain prevents a complementation of a CDC48 disruption, although this is the best conserved region between the two proteins. Protein chimeras containing the N-terminal part of VCP only complement a disruption of CDC48 when expressed at high levels. The respective yeast strain shows a nucleus devoid of Cdc48p. In contrast to VCP, Cdc48p contains an almost perfect nuclear targeting sequence in this region. Exchange of the C-terminal Cdc48p domain for the C-terminus of VCP leads to normal viability of the cell, even at low expression levels (Madeo, 1997).

Cdc48p from Saccharomyces cerevisiae and its highly conserved mammalian homolog VCP (valosin-containing protein) are ATPases with essential functions in cell division and homotypic fusion of endoplasmic reticulum vesicles. Both are mainly attached to the endoplasmic reticulum, but relocalize in a cell cycle-dependent manner: Cdc48p enters the nucleus during late G1; VCP aggregates at the centrosome during mitosis. The nuclear import signal sequence of Cdc48p was localized near the amino terminus and its function demonstrated by mutagenesis. The nuclear import is regulated by a cell cycle-dependent phosphorylation of a tyrosine residue near the carboxy terminus. Two-hybrid studies indicate that the phosphorylation results in a conformational change of the protein, exposing the nuclear import signal sequence previously masked by a stretch of acidic residues (Madeo, 1998).

Smid homologs are involved in the ubiquitin protein degradation pathway

A library of randomly generated 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. The gene involved, termed UFD3, encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrate that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Similar to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system opens up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics (Ghislain, 1996).

The inactivation of the prototype NF-kappaB inhibitor, IkappaBalpha, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. Valosin-containing protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, co-precipitates with IkappaBalpha immune complexes. The ubiquitinated IkappaBalpha conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-kappaB. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IkappaBalpha complexes sediment in the 19 S fractions, while the unmodified IkappaBalpha sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IkappaBalpha are critical for VCP binding, which in turn is necessary but not sufficient for IkappaBalpha degradation; while the N-terminal domain of IkappaBalpha is required in all three reactions, both N- and C-terminal domains are required in degradation. Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. These results suggest that VCP may provide a physical and functional link between IkappaBalpha and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IkappaBalpha (Dai, 1998).


REFERENCES

Search PubMed for articles about Drosophila Smallminded

Dai, R. M., et al. (1998). Involvement of valosin-containing protein, an ATPase Co-purified with IkappaBalpha and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IkappaBalpha. J. Biol. Chem. 273(6): 3562-73. PubMed ID: 9452483

Feiler, H. S., et al. (1995). The higher plant Arabidopsis thaliana encodes a functional CDC48 homologue which is highly expressed in dividing and expanding cells. EMBO J. 14(22): 5626-37. PubMed ID: 8521820

Frohlich, K. U., et al. (1991). Yeast cell cycle protein CDC48p shows full-length homology to the mammalian protein VCP and is a member of a protein family involved in xisome formation, and gene expression. J. Cell Biol. 114(3): 443-53. PubMed ID: 1860879

Ghislain, M., et al. (1996). Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae. EMBO J. 15(18): 4884-99. PubMed ID: 8890162

Latterich, M., Frohlich, K. U. and Schekman, R. (1995). Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes. Cell 82(6): 885-93. PubMed ID: 7553849

Long, A. R., et al. (1998a). Isolation and characterisation of smallminded, a Drosophila gene encoding a new member of the Cdc48p/VCP subfamily of AAA proteins. Gene 208(2): 191-9. PubMed ID: 9524263

Long, A. R., Wilkins, J. C. and Shepherd, D. (1998b). Dynamic developmental expression of smallminded, a Drosophila gene required for cell division. Mech. Dev. 76(1-2): 33-43. PubMed ID: 9767094

Madeo, F., Schlauer, J. and Frohlich, K. U. (1997). Identification of the regions of porcine VCP preventing its function in Saccharomyces cerevisiae. Gene 204(1-2): 145-51. PubMed ID: 9434177

Madeo, F., et al. (1998). Tyrosine phosphorylation regulates cell cycle-dependent nuclear localization of Cdc48p. Mol. Biol. Cell 9(1): 131-41. PubMed ID: 9436996

Pinter, M., et al. (1998). TER94, a Drosophila homolog of the membrane fusion protein CDC48/p97, is accumulated in nonproliferating cells: in the reproductive organs and in the brain of the imago. Insect Biochem. Mol. Biol. 28(2): 91-8. PubMed ID: 9639875

Rabouille, C., et al. (1995). An NSF-like ATPase, p97, and NSF mediate cisternal regrowth from mitotic Golgi fragments. Cell 82(6): 905-14. PubMed ID: 7553851

Wu, D., et al. (1999). C. elegans MAC-1, an essential member of the AAA family of ATPases, can bind CED-4 and prevent cell death. Development 126(9): 2021-2031. PubMed ID: 10101135


Biological Overview

date revised: 10 November 2017

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