Photoreception in Phytoplankton

In many species of phytoplankton, simple photoreceptors monitor ambient lighting. Photoreceptors provide a number of selective advantages including the ability to assess the time of day for circadian rhythms, seasonal changes, and the detection of excessive light intensities and harmful UV light. Photoreceptors also serve as depth gauges in the water column for behaviors such as diurnal vertical migration. Photoreceptors can be organized together with screening pigment into visible eyespots. In a wide variety of motile phytoplankton, including Chlamydomonas, Volvox, Euglena, and Kryptoperidinium, eyespots are light-sensitive organelles residing within the cell. Eyespots are composed of photoreceptor proteins and typically red to orange carotenoid screening pigments. This association of photosensory pigment with screening pigment allows for detection of light directionality, needed for light-guided behaviors such as positive and negative phototaxis. In Chlamydomonas, the eyespot is located in the chloroplast and Chlamydomonas expresses a number of photosensory pigments including the microbial channelrhodopsins (ChR1 and ChR2). Dinoflagellates are unicellular protists that are ecologically important constituents of the phytoplankton. They display a great deal of diversity in morphology, nutritional modes and symbioses, and can be photosynthetic or heterotrophic, feeding on smaller phytoplankton. Dinoflagellates, such as Kryptoperidinium foliaceum, have eyespots that are used for light-mediated tasks including phototaxis. Dinoflagellates belonging to the family Warnowiaceae have a more elaborate eye. Their eye-organelle, called an ocelloid, is a large, elaborate structure consisting of a focusing lens, highly ordered retinal membranes, and a shield of dark pigment. This complex eye-organelle is similar to multicellular camera eyes, such as our own. Unraveling the molecular makeup, structure and function of dinoflagellate eyes, as well as light-guided behaviors in phytoplankton can inform us about the selective forces that drove evolution in the important steps from light detection to vision. We show here that the evolution from simple photoreception to vision seems to have independently followed identical paths and principles in phytoplankton and animals, significantly strengthening our understanding of this important biological process

Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster.

As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the functions of glycosyl hydrolases in the secretory pathway

v7a14-hartman pgmkr

Purpose: To determine the protein and transcript levels for rhodopsin (Rh1), arrestin 1 (Arr1), and arrestin 2 (Arr2) over a 12 h light/12 h dark cycle in the retina of the fruit fly, Drosophila melanogaster. This information is important for understanding the process of photoreceptor membrane turnover. Methods: Drosophila were entrained for several generations to a daily 12 h light/12 h dark cycle. They were sacrificed at 4 h intervals, beginning at the time of onset of the light phase. Proteins were resolved by polyacrylamide gel electrophoresis (PAGE) and subjected to immunoblot analysis using antibodies directed to rhodopsin, NinaA, Arr1, and Arr2. Northern blots were incubated with riboprobes corresponding to the rhodopsin gene (ninaE), arrestin1(arr1), and arrestin2 (arr2). Results: In entrained Drosophila, protein and mRNA levels for rhodopsin, arrestin1, and arrestin2 were constant during a 12 h light/12 h dark cycle. Conclusions: These results indicate that rhodopsin and arrestin protein synthesis in Drosophila photoreceptors do not fluctuate on a daily cycle. These findings are similar to those obtained in Xenopus laevis, but in contrast to a variety of other vertebrate and invertebrate species

Pathway for Rh1 oligosaccharide trimming.

<p>Rh1 deglycosylation is a highly coordinated pathway that requires the sequential actions of a large number of glycosyl hydrolase (GH) enzymes and families. Here, we illustrate the major steps that are thought to occur during Rh1 deglycosylation, beginning with the precursor oligosaccharide, Glc₃Man₉GlcNAc₂. The enzymes responsible for equivalent hydrolysis reactions in mammals are shown above each arrow. The <i>Drosophila</i> enzymes predicted to perform these steps during Rh1 maturation are indicated below each arrow. Of particular note, the step mediated by <i>Drosophila</i> fused lobes is unique to insects and plants. Removal of this terminal GlcNAc residue has not been shown to occur in mammals. This explains why elongation of human glycoproteins leads to the formation of complex-type oligosaccharides, whereas elongation of <i>Drosophila</i> glycoproteins leads to the formation of pauci-mannosidic structures. Importantly, the subsequent steps contained within the grey box represent a novel pathway for Rh1 maturation. That is, the mammalian enzymes responsible for these cleavage events have only been characterized in the context of carbohydrate catabolism in the lysosome, but have no known roles in glycoprotein processing. Here, we propose that the corresponding enzymes in <i>Drosophila</i> play a critical role in Rh1 deglycosylation during biosynthesis, leading to the complete deglycosylation of Rh1. G (glucose), M (mannose), N (β-<i>N</i>-acetylglucosamine, GlcNAc), Man (mannosidase), Hexo (hexosaminidase).</p

<i>Drosophila</i> α-Man-II functions upstream from α-Man-IIb during Rh1 deglycosylation.

<p>Intron/Exon structures for (<b>A</b>) <i>α-mannosidase-II</i> (<i>α-man-II</i>, CG18802) and (<b>B</b>) <i>α-mannosidase-IIb</i> (<i>α-man-IIb</i>, CG4606) are shown to indicate the alleles used in this study: <i>P[EP]α-man-II^G4901</i>, <i>P[XP]α-man-II^d06422</i>, <i>Pbac[SAstopDsRed]α-man-II^LL01094</i>, <i>P[GD2875]^v5838</i>, <i>Pbac[PB]α-man-IIb^c06077</i>, <i>Pbac[RB]α-man-IIb^e01163</i>, <i>Pbac[WH]α-man-IIb^f03535</i>, <i>Pbac[WH]α-man-IIb^f02524</i>, and <i>α-man-IIb^G572E</i>. Blue = coding sequence, red = additional mRNA, yellow = RNAi target. (<b>C</b>) Western blots of Rh1 protein from <i>α-man-II</i> (Left) and <i>α-man-IIb</i> (Right) mutant flies. Left Lanes: (1) Wild-type (WT), (2) <i>α-man-II^LL01094</i>, (3) <i>α-man-II^v5838</i>, (4) <i>α-man-II^G4901</i>, (5) <i>α-man-II^d06422</i>, and (6) WT. Right Lanes: (1) WT, (2) <i>α-man-IIb^G572E</i>, (3) <i>α-man-IIb^f02524</i>, (4) <i>α-man-IIb^f03535</i>, (5) <i>α-man-IIb^e01163</i>, (6) <i>α-man-IIb^c06077</i>, and (7) WT. One head was loaded per lane, with the exception of lane 3 on the left side (<i>α-man-II^v5838</i>), in which 4 heads were loaded. (<b>D</b>) Western blot, comparing the molecular weight of Rh1 in the <i>α-man-II^LL01094</i> and <i>α-man-IIb^f02524</i> mutants, indicating that Rh1 is slightly larger in <i>α-man-II</i> mutants. One half of a head was loaded per lane. (<b>E and F</b>) Western blot of Rh1 protein from the <i>α-man-II</i> and <i>α-man-IIb</i> alleles described in (C), treated (+) with either Endo H or PNGase F enzyme and labeled for Rh1. (<b>G</b>) Proposed role for <i>Drosophila</i> α-Man-II in trimming the M7 and M8 mannose residues (blue) during Rh1 biosynthesis. Our data indicate that <i>Drosophila</i> α-Man-IIb functions downstream at a step that is distinct from α-Man-II during Rh1 deglycosylation.</p

<i>Drosophila</i> Hexo1 is essential for Rh1 deglycosylation.

<p>(<b>A</b>) Intron/Exon structures for <i>hexosaminidase1</i> (<i>hexo1</i>, CG1318), indicating the alleles used in this study: <i>Pbac[RB]hexo1^e00001</i> and <i>hexo1^Q592X</i>. Blue = coding sequence, red = additional mRNA. (<b>B</b>) Western blot of Rh1 protein from <i>hexo1</i> mutant flies. Lanes: (1) WT, (2), <i>hexo1^Q592X</i>^{/Df(3L)ED4341}, (3) <i>hexo1^e00001</i>, and (4) WT. Deficiency Df(3L)ED4341 deletes cytological region 63F6-64B9 and thus fails to complement the <i>hexo1</i> locus, which lies at 64A12. 2 heads were loaded per lane. (<b>C</b>) Western blot of Rh1 protein from the <i>hexo1</i> alleles described in (B), treated (+) with either Endo H or PNGase F enzyme. (<b>D</b>) Proposed role for <i>Drosophila</i> Hexo1 in trimming the N2 GlcNAc residue from the N-glycan during Rh1 biosynthesis.</p

N-glycan processing enzymes in humans and <i>Drosophila</i>.

<p>Human and <i>Drosophila</i> enzymes in numerous glycosyl hydrolase (GH) families are involved in the processing and/or catabolism of N-glycans. These proteins are divided into six major groups and are listed in the order in which they are thought to function in the cascade (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004349#pgen-1004349-g004" target="_blank">Figure 4</a>). (<b>1</b>) Glucosidase I and the α-subunit of glucosidase II are from GH Families 63 and 31, respectively (tan). The β-subunit of glucosidase II is not a GH enzyme (tan). (<b>2</b>) The Class I α-mannosidases from GH Family 47 can be classified into three functionally distinct subgroups: Subgroup A includes the ER α1,2-mannosidases (orange), Subgroup B includes the Golgi α1,2-mannosidases (yellow), and Subgroup C includes the EDEMs (green). (<b>3</b>) GlcNAc-transferase is not a GH enzyme (dark blue). (<b>4</b>) The Class II α-mannosidases from GH Family 38 can also be classified into several functionally distinct subgroups: Subgroup A includes the Golgi α1,3(1,6)-mannosidases (light blue), Subgroup B includes the lysosomal α-mannosidases (purple), and Subgroup C includes an ER/cytosolic α-mannosidase, which is not found in <i>Drosophila</i> (grey). (<b>5</b>) The β-mannosidases from GH Family 2 (pink) and (<b>6</b>) the hexosaminidases from GH Family 20 (brown) are also listed. Accession numbers presented here indicate the protein sequences that were used for all amino acid alignments and sequence analyses performed in this study. Man (mannosidase), Hexo (hexosaminidase), L (lysosomal).</p

A schematic of the asparagine (Asn, N)-linked oligosaccharide precursor, Glc₃Man₉GlcNAc₂.

<p>The N-glycan contains three glucose residues (triangles, G), nine mannose residues (circles, M3–11), and two β-<i>N</i>-acetylglucosamine residues (squares, N1–2). A, B, and C refer to the major branches on the N-glycan structure. Colors complement the enzyme list provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004349#pgen-1004349-g002" target="_blank">Figure 2</a>, indicating the glycosyl hydrolases (GH) responsible for trimming each residue. The α1,2-linked mannose residues (M6, M9, M10, and M11) are removed by Class I α-mannosidases (GH Family 47). The α1,3- and α1,6-linked mannose residues (M4, M5, M7, and M8) are removed by Class II α-mannosidases (GH Family 38). M3 is attached to the N-glycan by a β1,4-linkage and is removed by a β-mannosidase (GH Family 2). Finally, the terminal GlcNAc residue is attached via a β1,2-linkage and is removed by a β-hexosaminidase (GH Family 20). Man (mannosidase), Hexo (hexosaminidase).</p