Amino Acid Metabolic Origin as an Evolutionary Influence on Protein Sequence in Yeast

The metabolic cycle of Saccharomyces cerevisiae consists of alternating oxidative (respiration) and reductive (glycolysis) energy-yielding reactions. The intracellular concentrations of amino acid precursors generated by these reactions oscillate accordingly, attaining maximal concentration during the middle of their respective yeast metabolic cycle phases. Typically, the amino acids themselves are most abundant at the end of their precursor’s phase. We show that this metabolic cycling has likely biased the amino acid composition of proteins across the S. cerevisiae genome. In particular, we observed that the metabolic source of amino acids is the single most important source of variation in the amino acid compositions of functionally related proteins and that this signal appears only in (facultative) organisms using both oxidative and reductive metabolism. Periodically expressed proteins are enriched for amino acids generated in the preceding phase of the metabolic cycle. Proteins expressed during the oxidative phase contain more glycolysis-derived amino acids, whereas proteins expressed during the reductive phase contain more respiration-derived amino acids. Rare amino acids (e.g., tryptophan) are greatly overrepresented or underrepresented, relative to the proteomic average, in periodically expressed proteins, whereas common amino acids vary by a few percent. Genome-wide, we infer that 20,000 to 60,000 residues have been modified by this previously unappreciated pressure. This trend is strongest in ancient proteins, suggesting that oscillating endogenous amino acid availability exerted genome-wide selective pressure on protein sequences across evolutionary time

Reph, a Regulator of Eph Receptor Expression in the Drosophila melanogaster Optic Lobe

Receptors of the Eph family of tyrosine kinases and their Ephrin ligands are involved in developmental processes as diverse as angiogenesis, axon guidance and cell migration. However, our understanding of the Eph signaling pathway is incomplete, and could benefit from an analysis by genetic methods. To this end, we performed a genetic modifier screen for mutations that affect Eph signaling in Drosophila melanogaster. Several dozen loci were identified on the basis of their suppression or enhancement of an eye defect induced by the ectopic expression of Ephrin during development; many of these mutant loci were found to disrupt visual system development. One modifier locus, reph (regulator of eph expression), was characterized in molecular detail and found to encode a putative nuclear protein that interacts genetically with Eph signaling pathway mutations. Reph is an autonomous regulator of Eph receptor expression, required for the graded expression of Eph protein and the establishment of an optic lobe axonal topographic map. These results reveal a novel component of the regulatory pathway controlling expression of eph and identify reph as a novel factor in the developing visual system

A novel proteolytic event controls Hedgehog intracellular sorting and distribution to receptive fields

ABSTRACT The patterning activity of a morphogen depends on secretion and dispersal mechanisms that shape its distribution to the cells of a receptive field. In the case of the protein Hedgehog (Hh), these mechanisms of secretion and transmission remain unclear. In the developing Drosophila visual system, Hh is partitioned for release at opposite poles of photoreceptor neurons. Release into the retina regulates the progression of eye development; axon transport and release at axon termini trigger the development of postsynaptic neurons in the brain. Here we show that this binary targeting decision is controlled by a C-terminal proteolysis. Hh with an intact C-terminus undergoes axonal transport, whereas a C-terminal proteolysis enables Hh to remain in the retina, creating a balance between eye and brain development. Thus, we define a novel mechanism for the apical/basal targeting of this developmentally important protein and posit that similar post-translational regulation could underlie the polarity of related ligands

Analysis of axonal trafficking via a novel live-imaging technique reveals distinct hedgehog transport kinetics

ABSTRACT The Drosophila melanogaster (Dmel) eye is an ideal model to study development, intracellular signaling, behavior, and neurodegenerative disease. Interestingly, dynamic data are not commonly employed to investigate eye-specific disease models. Using axonal transport of the morphogen Hedgehog (Hh), which is integral to Dmel eye-brain development and implicated in stem cell maintenance and neoplastic disease, we demonstrate the ability to comprehensively quantify and characterize its trafficking in various neuron types and a neurodegeneration model in live early third-instar larval Drosophila. We find that neuronal Hh, whose kinetics have not been reported previously, favors fast anterograde transport and varies in speed and flux with respect to axonal position. This suggests distinct trafficking pathways along the axon. Lastly, we report abnormal transport of Hh in an accepted model of photoreceptor neurodegeneration. As a technical complement to existing eye-specific disease models, we demonstrate the ability to directly visualize transport in real time in intact and live animals and track secreted cargoes from the axon to their release points. Particle dynamics can now be precisely calculated and we posit that this method could be conveniently applied to characterizing disease pathogenesis and genetic screening in other established models of neurodegeneration

Endocytic pathway is required for Drosophila Toll innate immune signaling

The Toll signaling pathway is required for the innate immune response against fungi and Gram-positive bacteria in Drosophila. Here we show that the endosomal proteins Myopic (Mop) and Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) are required for the activation of the Toll signaling pathway. This requirement is observed in cultured cells and in flies, and epistasis experiments show that the Mop protein functions upstream of the MyD88 adaptor and the Pelle kinase. Mop and Hrs, which are critical components of the ESCRT-0 endocytosis complex, colocalize with the Toll receptor in endosomes. We conclude that endocytosis is required for the activation of the Toll signaling pathway

Isolation of <i>eph</i> and <i>ephrin</i> mutants.

<p>A) The 10.2 kb <i>eph</i> locus (topmost diagram) consists of 14 exons (boxes; white = UTRs, black = coding sequences) and 13 introns (lines), localized to the 102D2-D5 region of chromosome IV. The core donor construct (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037303#s2" target="_blank">Methods</a> for details) consisted of <i>eph</i> genomic sequences (blue) lacking exons 1–4 (deleting the 5′UTR, start codon, signal sequence and a portion of the exoplasmic domain) and a 3′ region lacking the kinase domain and terminal 3′ sequences. An <i>I-SceI</i> site was engineered into the middle of exon 6 (red shading). The core construct was placed upstream of a white gene marker (<i>w^hs</i>, green shading), the whole being bracketed by FLP recognition target sequences (FRT, purple shading) and inserted into the transformation vector. ‘Ends-in’ recombination induced by FLP and <i>I-SceI</i> resulted in partial tandem duplication of the <i>eph</i> locus (bottom-most diagram). B) Southern blot analysis of five candidate <i>eph^KD</i> targeting events. <u>Lane 1</u>: molecular weight markers. <u>Lanes 2–8</u>: <i>NotI</i> digests of genomic DNA derived from: L2 (Canton S, control), L3 (Donor line, control), L4 (<i>eph^KD</i>1), L5 (<i>eph^KD</i>2), L6 (<i>eph^KD</i>3), L7 (<i>eph^KD</i>4) and L8 (<i>eph^KD</i>5). The lower molecular weight band (non-mobilized donor construct) was absent from <i>eph^KD</i> lines 1, 2 & 4 indicating successful homologous recombination. <u>Lanes 9–15</u>: <i>BglII</i> digests of genomic DNA derived from: L9 (Canton S, control), L10 (Donor line, control), L11 (<i>eph^KD</i>1), L12 (<i>eph^KD</i>2), L13 (<i>eph^KD</i>3), L14 (<i>eph^KD</i>4) and L15 (<i>eph^KD</i>5). The convergence of distinct donor and endogenous <i>eph</i> bands into a single band due to homologous recombination is clearly evident in the <i>eph^KD</i>1, 2 & 4 lines (L11, L12 and L14). C) RT-PCR using primers for the full-length <i>eph</i> transcript (L1–9, left side of gel and left diagram; primer locations indicated by arrows). L1 (Canton S, control), L2 (Donor line, control), L3 (<i>eph^KD</i>1/<i>eph^KD</i>1), L4 (<i>eph^KD</i>1/+), L5 (<i>eph^KD</i>2/+), L6 (<i>eph^KD</i>2/<i>eph^KD</i>2), L7 (<i>eph^KD</i>4/+), L8 (<i>eph^KD</i>4/<i>eph^KD</i>4), L9 (<i>eph^KD</i>3/<i>eph^KD</i>3), L10 (molecular weight markers). Full-length transcript was not detected in <i>eph^KD</i> homozygous animals (26 cycles). RT-PCR using primers for the 3′-deleted isoform of Eph is shown in L11–L19, right side of gel and right diagram (primer locations indicated by arrows). Source RNA for L11–L19 was identical to L1–L9. Only the truncated Eph isoform was expressed in <i>eph^KD</i> animals. <u>Abbreviations</u>: LBD (ligand-binding domain), FNIII (fibronectin type III repeats), JXM (juxtamembrane region), TK (tyrosine kinase domain), SAM (sterile alpha motif), PDZ (postsynaptic density 95/Discs-large/zona occludens-1 domain). D,D′) Eph (anti-Eph, red in D, shown alone in D′) is expressed on cortical neuron axons in wild-type third instar larvae, accumulating in a high-midline low-dorsoventral gradient in the medulla neuropil (compare anti-HRP staining, green, to anti-Eph staining, red). E,E′) Lack of Eph immunoreactivity (anti-Eph, red in E, shown alone in E′) corresponded to optic lobe defects (anti-HRP, green) in <i>eph^KD</i> animals, manifest as gaps in the neuropil (arrowheads). F) Schematic of the 5.8 kb <i>ephrin</i> locus localized to the 102C2 region of chromosome IV. The <i>ephrin</i> gene is comprised of 5 exons (black boxes) and 4 introns (gray lines). The start codon (red arrow) and RS5 P-element insertion site (red shaded box) into 5′UTR of the first exon are also indicated. G,G′) In wild-type animals, Ephrin expression (anti-Ephrin, red in G, shown alone in G′) is punctate along cortical neuron axons and concentrated in the optic lobe neuropil (anti-HRP, green in G). H,H′) In <i>ephrin^RS5</i> mutants, Ephrin expression is considerably reduced in the optic lobe (anti-Ephrin, red in H, shown alone in H′), resulting in neuropil defects (arrowheads; anti-HRP, green in H). White or yellow bars indicate the dorsoventral midline. Scale bar in D is 20 µm for D,D′,E,E′, G,G′, H,H′.</p

Ectopic misexpression of <i>reph</i> up-regulates Eph expression in the developing visual system.

<p>To determine whether <i>reph⁺</i> was sufficient for Eph expression, the UAS, GAL4 system was used to drive expression of a <i>UAS-reph⁺</i> transgene in cell-specific patterns or within somatic clones. GAL4-expressing cells and clones were positively marked by membrane-bound GFP expressed from a <i>UAS-CD8::GFP</i> transgene (green color in B and C; shown alone in B′. The axonal architecture was visualized by staining with anti-HRP (green in A, red in C). A,A′,A″) A wild type specimen showing the medulla and its characteristic high midline-low dorsoventral gradient of Eph (anti-Eph, blue in A, shown alone in A″). B,B′,B″) Expression of <i>UAS-reph⁺</i> in cortical neurons using an <i>elav-GAL4</i> driver flattens the Eph gradient, notably at the dorsoventral margins (anti-Eph, blue in B, shown alone in B″). Defects in medulla development seen as gaps in the neuropil (yellow arrowheads in B′,B″) result from this up-regulation of Eph expression. C,C′,C″) Several <i>UAS-reph⁺</i> cortical clones (red arrowheads in C′,C″) generated using a flip-out <i>tubGAL4</i> driver can be seen in this specimen. Within these clones, Eph expression (anti-Eph, shown alone in C″) was up-regulated. The enhanced Eph expression was associated with defects manifest as large HRP⁺ cortical inclusions. Disruption of the normal Eph expression pattern also affected medulla neuropil development (yellow arrowhead in C′, C″). Abbreviations: lobula (lob), medulla neuropil (med n).</p

A genetic screen for Eph pathway signaling molecules.

<p>A) The wild-type adult eye displays a regular ommatidial lattice. B) The rough-eye phenotype generated by expression of <i>UAS-ephrin</i> under control of the <i>sevenless2-GAL4</i> driver (SE) used to screen for modifier mutations. C) Near-complete suppression of the SE phenotype by co-expression of a dominant-negative <i>eph</i> transgene (<i>eph^DN</i>). D) Suppression of the SE phenotype by the <i>reph¹</i> allele. E) Suppression of the SE phenotype by <i>reph^k8617</i>. F) Co-expression of a <i>UAS-reph⁺</i> transgene enhances the SE phenotype. All images (20×) were acquired through a digital camera attached to a Zeiss Stemi SV11 stereo dissecting microscope. Images were captured using FPG3 software.</p