Regulation of SREBP processing and membrane lipid production by phospholipids in Drosophila.

Animal cells exert exquisite control over the physical and chemical properties of their membranes, but the mechanisms are obscure. We show that phosphatidylethanolamine, the major phospholipid in Drosophila, controls the release of sterol regulatory element-binding protein (SREBP) from Drosophila cell membranes, exerting feedback control on the synthesis of fatty acids and phospholipids. The finding that SREBP processing is controlled by different lipids in mammals and flies (sterols and phosphatidylethanolamine, respectively) suggests that an essential function of SREBP is to monitor cell membrane composition and to adjust lipid synthesis accordingly.

Molecular identification of the sterol-regulated luminal protease that cleaves SREBPs and controls lipid composition of animal cells.

The lipid composition of animal cells is controlled by SREBPs, transcription factors released from membranes by sterol-regulated proteolysis. Release is initiated by Site-1 protease (S1P), which cleaves SREBPs in the ER luminal loop between two membrane-spanning regions. To clone S1P, we prepared pCMV-PLAP-BP2, which encodes a fusion protein that contains placental alkaline phosphatase (PLAP) in the ER lumen flanked by cleavage sites for signal peptidase and S1P. In sterol-deprived cells, cleavage by both proteases leads to PLAP secretion. PLAP is not secreted by SRD-12B cells, cholesterol auxotrophs that lack S1P. We transfected SRD-12B cells with pCMV-PLAP-BP2 plus pools of CHO cDNAs and identified a cDNA that restores Site-1 cleavage and PLAP secretion. The cDNA encodes S1P, an intraluminal 1052-amino-acid membrane-bound subtilisin-like protease. We propose that S1P is the sterol-regulated protease that controls lipid metabolism in animal cells.

A short internal eliminated sequence with central conserved sequences interrupting the LA-MSC gene of the 81 locus in the hypotrichous ciliates Oxytricha fallax and O. trifallax.

IES-LA is a short Internal Eliminated Sequence interrupting LA-MSC, a protein-coding gene of the 81 locus of Oxytricha fallax and O. trifallax. IES-LA is precisely excised from the gene during development of the macronucleus. The internal eliminated sequence is bounded by CAAT ... AATG, and thereby resembles a TBE1 transposon internal eliminated sequence insertion that is grossly shortened (4.1 kbp to 52-64 bp), consistent with the hypothesis that short IESs are degenerated ancient transposons. The pattern of sequence conservation between five alleles of IES-LA shows that it differs from previously characterized classes of ciliate short IESs: while many short IESs have conserved ends and diverged centers, IES-LA is more conserved in its center and its ends are diverged. This implies a excision mechanism for IES-LA that is distinct from those for other known Oxytricha IESs.

Selection on the protein-coding genes of the TBE1 family of transposable elements in the ciliates Oxytricha fallax and O. trifallax.

TBE1s are "cut-and-paste" transposable elements found in high copy number in the germline genomes of the ciliates Oxytricha fallax and O. trifallax. TBE1 "family" sequence (sequence of mixed polymerase chain reaction products generated using primers that match roughly half the TBE1s in host whole-cell DNA) was obtained from both host species. Although family sequence autoradiograms represent thousands of different elements, they are as legible as those representing corresponding sequences of a single TBE1, implying that ideal polymorphisms are rare within the genes examined. Nucleotide polymorphisms among TBE1s (indicated by ambiguities in family sequence) are far more common at third than at first or second positions of codons of genes, implying that selection has conserved the amino acid sequences of these genes in the majority of TBE1s. Portions of the transposase gene and another TBE1 gene have been sequenced from 10 individual TBE1s. None of these portions is interrupted by stop codons or frameshifts, and, for both genes, pairwise comparisons of these sequences show that nonsynonymous differences are significantly less common than synonymous differences, again implicating conservative selection Phylogenetic analysis shows that multiple divergent lineages of TBE1s have evolved under this selection within O. fallax. All these results are unexpected for cut-and-paste transposons in eukaryotic hosts: since transposase encoded by intact elements presumably acts in trans, it can duplicate mutant copies (those that do not encode functional transposase) found in the same genome, and thus no selection is expected to maintain the transposase gene. The selection demonstrated here could act at transposition (if functional TBE1s are preferentially transposed) or at the level of the host (if the host's fitness depends on functional TBE1 genes). TBE1-encoded proteins might be responsible for the precise excision of TBE1s that occurs during development of the host somatic nucleus; selection on hosts for uninterrupted somatic genes would then translate into selection for TBE1 protein-coding competence. We suggest a method for distinguishing between these two classes of explanations by finding and analyzing divergent alleles of ancestral transposable element insertions.

Two two-gene macronuclear chromosomes of the hypotrichous ciliates Oxytricha fallax and O. trifallax generated by alternative processing of the 81 locus.

We describe the first know macronuclear chromosomes that carry more than one gene in hypotrichous ciliated protozoa. These 4.9- and 2.8-kbp chromosomes each consist almost exclusively of two protein-coding genes, which are conserved and transcribed. The two chromosomes share a common region that consists of a gene that is a member of the family of mitochondrial solute carrier genes (CR-MSC; [Williams and Herrick (1991): Nucleic Acids Res 19:4717-4724]. Each chromosome also carries another gene appended to its common region: The 4.9-kbp chromosome also carries a gene that encodes a protein that is rich in glutamine and charged amino acids and bears regions of heptad repeats characteristic of coiled-coils. Its function is unknown. The second gene of the 2.8 kbp chromosome is a mitochondrial solute carrier gene (LA-MSC); thus, the 2.8-kbp chromosomes consists of two mitochondrial solute carrier paralogs. Phylogenetic analysis indicates that the two genes were duplicated before ciliates diverged from the main eukaryotic lineage and were subsequently juxtaposed. The CR- and LA-MSC genes are each interrupted by three introns. The introns are not in homologous positions, suggesting that they may have originated from multiple group II intron transpositions. These chromosomes and their genes are encoded in the Oxytricha germline by the 81 locus. This locus is alternatively processed to generate a nested set of three macronuclear chromosomes, the 4.9- and 2.8-kbp chromosomes and a third (1.6 kbp) which consists almost exclusively of the shared common gene, CR-MSC. Such alternative processing is common in macronuclear development of O. fallax [Cartinhour and Herrick (1984): Mol Cell Biol 4:931-938]. Possible functions for alternative processing are considered; e.g., it may serve to physically link genes to allow co-regulation or co-replication by a common cis-acting sequence.

Internal eliminated sequences interrupting the Oxytricha 81 locus: allelic divergence, conservation, conversions, and possible transposon origins.

Internal eliminated sequences (IESs) often interrupt ciliate genes in the silent germline nucleus but are exactly excised and eliminated from the developing somatic nucleus from which genes are then expressed. Some long IESs are transposons, supporting the hypothesis that short IESs are ancient transposon relics. In light of that hypothesis and to explore the evolutionary history of a collection of IESs, we have compared various alleles of a particular locus (the 81 locus) of the ciliated protozoa Oxytricha trifallax and O. fallax. Three short IESs that interrupt two genes of the locus are found in alleles from both species, and thus must be relatively ancient, consistent with the hypothesis that short IESs are transposon relics. In contrast, TBE1 transposon interruptions of the locus are allele-specific and probably the results of recent transpositions. These IESs (and the TBE1s) are precisely excised from the DNA of the developing somatic macronucleus. Each IES interrupts a highly conserved sequence. A few nucleotides at the ends of each IES are also conserved, suggesting that they interact critically with IES excision machinery. However, most IES nucleotide positions have evolved at high rates, showing little or no selective constraint for function. Nonetheless, the length of each IES has been maintained (+/- 3 bp). While one IES is approximately 33 bp long, three other IESs have very similar sizes, approximately 70 bp long. Two IESs are surrounded by direct repeats of the sequence TTCTT. No other sequence similarities were found between any of the four IESs. However, the ends of one IES do match the inverted terminal repeat consensus sequence of the "TA" IESs of Paramecium. Three O. trifallax alleles appear to have been recipients in recent conversion events that could have been provoked by double-strand breaks associated with IES ends subsequent to IES transposition. Our findings support the hypothesis that short IESs evolved from ancient transposons that have lost most of their sequences, except those necessary for precise excision during macronuclear development.