RESUMEN
Volvocine green algae are a model for understanding the evolution of mating types and sexes. They are facultatively sexual, with gametic differentiation occurring in response to nitrogen starvation (-N) in most genera and to sex inducer hormone in Volvox. The conserved RWP-RK family transcription factor (TF) MID is encoded by the minus mating-type locus or male sex-determining region of heterothallic volvocine species and dominantly determines minus or male gametic differentiation. However, the factor(s) responsible for establishing the default plus or female differentiation programs have remained elusive. We performed a phylo-transcriptomic screen for autosomal RWP-RK TFs induced during gametogenesis in unicellular isogamous Chlamydomonas reinhardtii (Chlamydomonas) and in multicellular oogamous Volvox carteri (Volvox) and identified a single conserved ortho-group we named Volvocine Sex Regulator 1 (VSR1). Chlamydomonas vsr1 mutants of either mating type failed to mate and could not induce expression of key mating-type-specific genes. Similarly, Volvox vsr1 mutants in either sex could initiate sexual embryogenesis, but the presumptive eggs or androgonidia (sperm packet precursors) were infertile and unable to express key sex-specific genes. Yeast two-hybrid assays identified a conserved domain in VSR1 capable of self-interaction or interaction with the conserved N terminal domain of MID. In vivo coimmunoprecipitation experiments demonstrated association of VSR1 and MID in both Chlamydomonas and Volvox. These data support a new model for volvocine sexual differentiation where VSR1 homodimers activate expression of plus/female gamete-specific-genes, but when MID is present, MID-VSR1 heterodimers are preferentially formed and activate minus/male gamete-specific-genes.
Asunto(s)
Chlamydomonas , Semillas , Sexo , Reproducción , Células Germinativas , Espermatozoides , BiotinaRESUMEN
Multicellularity is the premier example of a major evolutionary transition in individuality and was a foundational event in the evolution of macroscopic biodiversity. The volvocine chlorophyte lineage is well suited for studying this process. Extant members span unicellular, simple colonial, and obligate multicellular taxa with germ-soma differentiation. Here, we report the nuclear genome sequence of one of the most morphologically simple organisms in this lineage-the 4-celled colonial Tetrabaena socialis and compare this to the three other complete volvocine nuclear genomes. Using conservative estimates of gene family expansions a minimal set of expanded gene families was identified that associate with the origin of multicellularity. These families are rich in genes related to developmental processes. A subset of these families is lineage specific, which suggests that at a genomic level the evolution of multicellularity also includes lineage-specific molecular developments. Multiple points of evidence associate modifications to the ubiquitin proteasomal pathway (UPP) with the beginning of coloniality. Genes undergoing positive or accelerating selection in the multicellular volvocines were found to be enriched in components of the UPP and gene families gained at the origin of multicellularity include components of the UPP. A defining feature of colonial/multicellular life cycles is the genetic control of cell number. The genomic data presented here, which includes diversification of cell cycle genes and modifications to the UPP, align the genetic components with the evolution of this trait.
Asunto(s)
Evolución Biológica , Chlorophyta/genética , Genes cdc , Componentes Genómicos , Ciclinas/genética , Genes de Retinoblastoma , Familia de Multigenes , Complejo de la Endopetidasa Proteasomal/genética , Selección Genética , Transcriptoma , Ubiquitina/genéticaRESUMEN
Heteromorphic sex-determining regions or mating-type loci can contain large regions of non-recombining sequence where selection operates under different constraints than in freely recombining autosomal regions. Detailed studies of these non-recombining regions can provide insights into how genes are gained and lost, and how genetic isolation is maintained between mating haplotypes or sex chromosomes. The Chlamydomonas reinhardtii mating-type locus (MT) is a complex polygenic region characterized by sequence rearrangements and suppressed recombination between its two haplotypes, MT+ and MT-. We used new sequence information to redefine the genetic contents of MT and found repeated translocations from autosomes as well as sexually controlled expression patterns for several newly identified genes. We examined sequence diversity of MT genes from wild isolates of C. reinhardtii to investigate the impacts of recombination suppression. Our population data revealed two previously unreported types of genetic exchange in Chlamydomonas MT--gene conversion in the rearranged domains, and crossover exchanges in flanking domains--both of which contribute to maintenance of genetic homogeneity between haplotypes. To investigate the cause of blocked recombination in MT we assessed recombination rates in crosses where the parents were homozygous at MT. While normal recombination was restored in MT+ ×MT+ crosses, it was still suppressed in MT- ×MT- crosses. These data revealed an underlying asymmetry in the two MT haplotypes and suggest that sequence rearrangements are insufficient to fully account for recombination suppression. Together our findings reveal new evolutionary dynamics for mating loci and have implications for the evolution of heteromorphic sex chromosomes and other non-recombining genomic regions.
Asunto(s)
Chlamydomonas reinhardtii/genética , Sitios Genéticos/genética , Recombinación Genética , Reproducción/genética , Cromosomas Sexuales/genética , Translocación Genética/genética , Evolución Biológica , Chlamydomonas reinhardtii/crecimiento & desarrollo , Conversión Génica , HaplotiposRESUMEN
It has been argued that for certain lineages, noncoding DNA expansion is a consequence of the increased random genetic drift associated with long-term escalations in organism size. But a lack of data has prevented the investigation of this hypothesis in most plastid-bearing protists. Here, using newly sequenced mitochondrial and plastid genomes, we explore the relationship between organelle DNA noncoding content and organism size within volvocine green algae. By looking at unicellular, colonial, and differentiated multicellular algae, we show that organelle DNA complexity scales positively with species size and cell number across the volvocine lineage. Moreover, silent-site genetic diversity data suggest that the volvocine species with the largest cell numbers and most bloated organelle genomes have the smallest effective population sizes. Together, these findings support the view that nonadaptive processes, like random genetic drift, promote the expansion of noncoding regions in organelle genomes.
Asunto(s)
Chlamydomonas reinhardtii/genética , Genoma Mitocondrial , Genoma de Plastidios , Mitocondrias/genética , Plastidios/genética , Chlamydomonas reinhardtii/citología , Evolución Molecular , Flujo Genético , Variación Genética , Genoma de Planta , Modelos Genéticos , Volvocida/citología , Volvocida/genéticaRESUMEN
The evolution of germ-soma cellular differentiation represents a key step in the evolution of multicellular individuality. Volvox carteri and its relatives, the volvocine green algae, provide a model system for studying the evolution of cellular differentiation. In V. carteri, the regA gene controls somatic cell differentiation and is found in a group of paralogs called the reg cluster, along with rlsA, rlsB, and rlsC. However, the developmental program of V. carteri is derived compared to other volvocine algae. Here we examine Volvox powersii which possesses an ancestral developmental program and independent evolution of the Volvox body plan. We sequenced the reg cluster from V. powersii wild-type and a mutant with fewer cells and altered germ-soma ratio. We found that the mutant strain's rlsB gene has a deletion predicted to cause a truncated protein product. We developed a genetic transformation procedure to insert wild-type rlsB into the mutant strain. Transformation did not result in phenotypic rescue, suggesting the rlsB mutation is insufficient for generating the mutant phenotype. The transformation techniques and sequences described here provide essential tools to study V. powersii, a species well suited for studying the evolution of cellular differentiation and convergent evolution of Volvox morphology.
Asunto(s)
Chlorophyta , Volvox , Secuencia de Bases , Diferenciación Celular , Volvox/genéticaRESUMEN
The evolution of anisogamy/oogamy in the colonial Volvocales might have occurred in an ancestral isogamous colonial organism like Gonium pectorale. The unicellular, close relative Chlamydomonas reinhardtii has a mating-type (MT) locus harboring several mating-type-specific genes, including one involved in mating-type determination and another involved in the function of the tubular mating structure in only one of the two isogametes. In this study, as the first step in identifying the G. pectorale MT locus, we isolated from G. pectorale the ortholog of the C. reinhardtii mating-type-determining minus-dominance (CrMID) gene, which is localized only in the MT- locus. 3'- and 5'-RACE RT-PCR using degenerate primers identified a CrMID-orthologous 164-amino-acid coding gene (GpMID) containing a leucine-zipper RWP-RK domain near the C-terminal, as is the case with CrMID. Genomic Southern blot analysis showed that GpMID was coded only in the minus strain of G. pectorale. RT-PCR revealed that GpMID expression increased during nitrogen starvation. Analysis of F1 progeny suggested that GpMID and isopropylmalate dehydratase LEU1S are tightly linked, suggesting that they are harbored in a chromosomal region under recombinational suppression that is comparable to the C. reinhardtii MT locus. However, two other genes present in the C. reinhardtii MT locus are not linked to the G. pectorale LEU1S/MID, suggesting that the gene content of the volvocalean MT loci is not static over time. Inheritance of chloroplast and mitochondria genomes in G. pectorale is uniparental from the plus and minus parents, respectively, as is also the case in C. reinhardtii.
Asunto(s)
Chlorophyta/genética , Genes Dominantes , Homología de Secuencia de Ácido Nucleico , Proteínas Algáceas/química , Proteínas Algáceas/genética , Alelos , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Secuencia Conservada , Exones/genética , Regulación de la Expresión Génica , Marcadores Genéticos , Patrón de Herencia/genética , Intrones/genética , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Reproducción , Alineación de SecuenciaRESUMEN
Volvox is a very interesting oogamous organism that exhibits various types of sexuality and/or sexual spheroids depending upon species or strains. However, molecular bases of such sexual reproduction characteristics have not been studied in this genus. In the model species V. carteri, an ortholog of the minus mating type-determining or minus dominance gene (MID) of isogamous Chlamydomonas reinhardtii is male-specific and determines the sperm formation. Male and female genders are genetically determined (heterothallism) in V. carteri, whereas in several other species of Volvox both male and female gametes (sperm and eggs) are formed within the same clonal culture (homothallism). To resolve the molecular basis of the evolution of Volvox species with monoecious spheroids, we here describe a MID ortholog in the homothallic species V. africanus that produces both monoecious and male spheroids within a single clonal culture. Comparison of synonymous and nonsynonymous nucleotide substitutions in MID genes between V. africanus and heterothallic volvocacean species suggests that the MID gene of V. africanus evolved under the same degree of functional constraint as those of the heterothallic species. Based on semi quantitative reverse transcription polymerase chain reaction analyses using the asexual, male and monoecious spheroids isolated from a sexually induced V. africanus culture, the MID mRNA level was significantly upregulated in the male spheroids, but suppressed in the monoecious spheroids. These results suggest that the monoecious spheroid-specific down regulation of gene expression of the MID homolog correlates with the formation of both eggs and sperm in the same spheroid in V. africanus.
Asunto(s)
Evolución Molecular , Genes de Plantas , Polen , Esferoides Celulares , Volvox/genética , Southern Blotting , Óvulo Vegetal , Filogenia , Reacción en Cadena de la Polimerasa , Reproducción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Volvox/clasificación , Volvox/fisiologíaRESUMEN
Sex-determining regions (SDRs) or mating-type (MT) loci in two sequenced volvocine algal species, Chlamydomonas reinhardtii and Volvox carteri, exhibit major differences in size, structure, gene content, and gametolog differentiation. Understanding the origin of these differences requires investigation of MT loci from related species. Here, we determined the sequences of the minus and plus MT haplotypes of the isogamous 16-celled volvocine alga, Gonium pectorale, which is more closely related to the multicellular V. carteri than to C. reinhardtii Compared to C. reinhardtii MT, G. pectorale MT is moderately larger in size, and has a less complex structure, with only two major syntenic blocs of collinear gametologs. However, the gametolog content of G. pectorale MT has more overlap with that of V. carteri MT than with C. reinhardtii MT, while the allelic divergence between gametologs in G. pectorale is even lower than that in C. reinhardtii Three key sex-related genes are conserved in G. pectorale MT: GpMID and GpMTD1 in MT-, and GpFUS1 in MT+. GpFUS1 protein exhibited specific localization at the plus-gametic mating structure, indicating a conserved function in fertilization. Our results suggest that the G. pectorale-V. carteri common ancestral MT experienced at least one major reformation after the split from C. reinhardtii, and that the V. carteri ancestral MT underwent a subsequent expansion and loss of recombination after the divergence from G. pectorale These data begin to polarize important changes that occurred in volvocine MT loci, and highlight the potential for discontinuous and dynamic evolution in SDRs.
Asunto(s)
Haplotipos , Sitios de Carácter Cuantitativo , Reproducción/genética , Volvox/genética , Paseo de Cromosoma , Biología Computacional , Evolución Molecular , Expresión Génica , Ligamiento Genético , Genoma de Planta , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Filogenia , Procesos de Determinación del Sexo/genética , Volvox/clasificaciónRESUMEN
The transition to multicellularity has occurred numerous times in all domains of life, yet its initial steps are poorly understood. The volvocine green algae are a tractable system for understanding the genetic basis of multicellularity including the initial formation of cooperative cell groups. Here we report the genome sequence of the undifferentiated colonial alga, Gonium pectorale, where group formation evolved by co-option of the retinoblastoma cell cycle regulatory pathway. Significantly, expression of the Gonium retinoblastoma cell cycle regulator in unicellular Chlamydomonas causes it to become colonial. The presence of these changes in undifferentiated Gonium indicates extensive group-level adaptation during the initial step in the evolution of multicellularity. These results emphasize an early and formative step in the evolution of multicellularity, the evolution of cell cycle regulation, one that may shed light on the evolutionary history of other multicellular innovations and evolutionary transitions.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Chlamydomonas/genética , Chlorophyta/genética , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Evolución Biológica , Chlamydomonas/citología , Chlorophyta/clasificación , Chlorophyta/citología , Tamaño del Genoma , Filogenia , Células Vegetales/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Transformación GenéticaRESUMEN
Portions of the cloned mating-type (MT) loci (mt(+) and mt(-)) of Chlamydomonas reinhardtii, defined as the approximately 1-Mb domains of linkage group VI that are under recombinational suppression, were subjected to Northern analysis to elucidate their coding capacity. The four central rearranged segments of the loci were found to contain both housekeeping genes (expressed during several life-cycle stages) and mating-related genes, while the sequences unique to mt(+) or mt(-) carried genes expressed only in the gametic or zygotic phases of the life cycle. One of these genes, Mtd1, is a candidate participant in gametic cell fusion; two others, Mta1 and Ezy2, are candidate participants in the uniparental inheritance of chloroplast DNA. The identified housekeeping genes include Pdk, encoding pyruvate dehydrogenase kinase, and GdcH, encoding glycine decarboxylase complex subunit H. Unusual genetic configurations include three genes whose sequences overlap, one gene that has inserted into the coding region of another, several genes that have been inactivated by rearrangements in the region, and genes that have undergone tandem duplication. This report extends our original conclusion that the MT locus has incurred high levels of mutational change.
Asunto(s)
Chlamydomonas reinhardtii/genética , Genes Protozoarios , Aminoácido Oxidorreductasas/genética , Animales , Northern Blotting , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/fisiología , Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Reordenamiento Génico , Complejo Glicina-Descarboxilasa , Glicina-Deshidrogenasa (Descarboxilante) , Datos de Secuencia Molecular , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Transcripción GenéticaRESUMEN
To understand the hierarchy of life in evolutionary terms, we must explain why groups of one kind of individual, say cells, evolve into a new higher level individual, a multicellular organism. A fundamental step in this process is the division of labor into nonreproductive altruistic soma. The regA gene is critical for somatic differentiation in Volvox carteri, a multicellular species of volvocine algae. We report the sequence of regA-like genes and several syntenic markers from divergent species of Volvox. We show that regA evolved early in the volvocines and predict that lineages with and without soma descended from a regA-containing ancestor. We hypothesize an alternate evolutionary history of regA than the prevailing "proto-regA" hypothesis. The variation in presence of soma may be explained by multiple lineages independently evolving soma utilizing regA or alternate genetic pathways. Our prediction that the genetic basis for soma exists in species without somatic cells raises a number of questions, most fundamentally, under what conditions would species with the genetic potential for soma, and hence greater individuality, not evolve these traits. We conclude that the evolution of individuality in the volvocine algae is more complicated and labile than previously appreciated on theoretical grounds.
Asunto(s)
Evolución Molecular , Duplicación de Gen , Genes de Plantas , Volvox/genética , Diferenciación Celular , Filogenia , Volvox/citologíaRESUMEN
BACKGROUND: Isogamous organisms lack obvious cytological differences in the gametes of the two complementary mating types. Consequently, it is difficult to ascertain which of the two mating types are homologous when comparing related but sexual isolated strains or species. The colonial volvocalean algal genus Gonium consists of such isogamous organisms with heterothallic mating types designated arbitrarily as plus or minus in addition to homothallic strains. Homologous molecular markers among lineages may provide an "objective" framework to assign heterothallic mating types. METHODOLOGY/PRINCIPAL FINDINGS: Using degenerate primers designed based on previously reported MID orthologs, the "master regulator" of mating types/sexes in the colonial Volvocales, MID homologs were identified and their presence/absence was examined in nine strains of four species of Gonium. Only one of the two complementary mating types in each of the four heterothallic species has a MID homolog. In addition to heterothallic strains, a homothallic strain of G. multicoccum has MID. Molecular evolutionary analysis suggests that MID of this homothallic strain retains functional constraint comparable to that of the heterothallic strains. CONCLUSION/SIGNIFICANCE: We coordinated mating genotypes based on presence or absence of a MID homolog, respectively, in heterothallic species. This scheme should be applicable to heterothallic species of other isogamous colonial Volvocales including Pandorina and Yamagishiella. Homothallism emerged polyphyletically in the colonial Volvocales, although its mechanism remains unknown. Our identification of a MID homolog for a homothallic strain of G. multicoccum suggests a MID-dependent mechanism is involved in the sexual developmental program of this homothallic species.
Asunto(s)
Chlorophyta/metabolismo , Animales , Chlorophyta/clasificación , Chlorophyta/genética , Femenino , Genotipo , Masculino , FilogeniaRESUMEN
Gametes were induced separately in cultures of each mating type of the heterothallic, isogamous colonial volvocalean Gonium pectorale O. F. Müll. to examine the tubular mating structure (TMS) of both mating types plus and minus (plus and minus), referred to as "bilateral mating papillae." Addition of dibutyryl cyclic adenosine monophosphate (DcAMP or db-cAMP) and 3-isobutyl-1-methylxanthine (IBMX) to approximately 3-week-old cultures of each mating type induced immediate release of naked gametes from the cell walls. Both plus and minus gametes formed a TMS in the anterior region of the protoplasts. Accumulation of actin was visualized by antibody staining in the TMS of both mating types as occurs in the TMS (fertilization tubule) of the plus gametes of the unicellular volvocalean Chlamydomonas reinhardtii P. A. Dang. Induction of naked gametes with a TMS in each mating type will be useful for future cell biological and evolutionary studies of the isogametes of colonial volvocalean algae.
RESUMEN
Although dimorphic sexes have evolved repeatedly in multicellular eukaryotes, their origins are unknown. The mating locus (MT) of the sexually dimorphic multicellular green alga Volvox carteri specifies the production of eggs and sperm and has undergone a remarkable expansion and divergence relative to MT from Chlamydomonas reinhardtii, which is a closely related unicellular species that has equal-sized gametes. Transcriptome analysis revealed a rewired gametic expression program for Volvox MT genes relative to Chlamydomonas and identified multiple gender-specific and sex-regulated transcripts. The retinoblastoma tumor suppressor homolog MAT3 is a Volvox MT gene that displays sexually regulated alternative splicing and evidence of gender-specific selection, both of which are indicative of cooption into the sexual cycle. Thus, sex-determining loci affect the evolution of both sex-related and non-sex-related genes.
Asunto(s)
Proteínas Algáceas/genética , Evolución Molecular , Genes , Sitios Genéticos , Volvox/genética , Volvox/fisiología , Proteínas Algáceas/metabolismo , Empalme Alternativo , División Celular , Chlamydomonas/genética , Chlamydomonas/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genes de Retinoblastoma , Intrones , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Recombinación Genética , Reproducción , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Análisis de Secuencia de ADNRESUMEN
The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are well suited for the investigation of the evolution of multicellularity and development. We sequenced the 138-mega-base pair genome of V. carteri and compared its approximately 14,500 predicted proteins to those of its unicellular relative Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials and few species-specific protein-coding gene predictions. Volvox is enriched in volvocine-algal-specific proteins, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity.
Asunto(s)
Proteínas Algáceas/química , Proteínas Algáceas/genética , Chlamydomonas reinhardtii/genética , Genoma , Volvox/genética , Proteínas Algáceas/metabolismo , Evolución Biológica , Chlamydomonas reinhardtii/citología , Chlamydomonas reinhardtii/crecimiento & desarrollo , Chlamydomonas reinhardtii/fisiología , ADN de Algas/genética , Evolución Molecular , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Genes , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Especificidad de la Especie , Sintenía , Volvox/citología , Volvox/crecimiento & desarrollo , Volvox/fisiologíaRESUMEN
Gonium pectorale O. F. Müll. (Volvocales, Chlorophyta), a colonial 8- or 16-cellular alga, is phylogenetically important as an intermediate form between isogametic unicellular Chlamydomonas and oogamous Volvox. We identified the mating-type specific gene GpMTD1, from G. pectorale, the first homologue of Chlamydomonas reinhardtii MTD1 (CrMTD1). The GpMTD1 gene was found to be present only in the minus mating-type locus and was expressed specifically in the gametic phase as is the case for CrMTD1, suggested to participate in development of the minus gametes. This gene is useful as a probe in analyzing the bacterial artificial chromosome (BAC) library for resolving genomic structures of the mating-type loci in isogamous and oogamous colonial volvocaleans.
RESUMEN
Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.
Asunto(s)
Proteínas Algáceas/genética , Proteínas Algáceas/fisiología , Evolución Biológica , Chlamydomonas reinhardtii/genética , Genoma , Animales , Chlamydomonas reinhardtii/fisiología , Cloroplastos/metabolismo , Biología Computacional , ADN de Algas/genética , Flagelos/metabolismo , Genes , Genómica , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Datos de Secuencia Molecular , Familia de Multigenes , Fotosíntesis/genética , Filogenia , Plantas/genética , Proteoma , Análisis de Secuencia de ADNRESUMEN
Gametes of the unicellular green alga Chlamydomonas reinhardtii undergo sexual adhesion via enormous chimeric Hyp-rich glycoproteins (HRGPs), the plus and minus sexual agglutinins, that are displayed on their flagellar membrane surfaces. We have previously purified the agglutinins and analyzed their structural organization using electron microscopy. We report here the cloning and sequencing of the Sag1 and Sad1 genes that encode the two agglutinins and relate their derived amino acid sequences and predicted secondary structure to the morphology of the purified proteins. Both agglutinin proteins are organized into three distinct domains: a head, a shaft in a polyproline II configuration, and an N-terminal domain. The plus and minus heads are related in overall organization but poorly conserved in sequence except for two regions of predicted hydrophobic alpha-helix. The shafts contain numerous repeats of the PPSPX motif previously identified in Gp1, a cell wall HRGP. We propose that the head domains engage in autolectin associations with the distal termini of their own shafts and suggest ways that adhesion may involve head-head interactions, exolectin interactions between the heads and shafts of opposite type, and antiparallel shaft-shaft interactions mediated by carbohydrates displayed in polyproline II configurations.
Asunto(s)
Aglutininas/genética , Chlamydomonas reinhardtii/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Clonación Molecular , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The chloroplast Albino3 (Alb3) protein is a chloroplast homolog of the mitochondrial Oxa1p and YidC proteins of Escherichia coli, which are essential components for integrating membrane proteins. In vitro studies in vascular plants have revealed that Alb3 is required for the integration of the light-harvesting complex protein into the thylakoid membrane. Here, we show that the gene affected in the ac29 mutant of Chlamydomonas reinhardtii is Alb3.1. The availability of the ac29 mutant has allowed us to examine the function of Alb3.1 in vivo. The loss of Alb3.1 has two major effects. First, the amount of light-harvesting complex from photosystem II (LHCII) and photosystem I (LHCI) is reduced >10-fold, and total chlorophyll represents only 30% of wild-type levels. Second, the amount of photosystem II is diminished 2-fold in light-grown cells and nearly 10-fold in dark-grown cells. The accumulation of photosystem I, the cytochrome b(6)f complex, and ATP synthase is not affected in the ac29 mutant. Mild solubilization of thylakoid membranes reveals that Alb3 forms two distinct complexes, a lower molecular mass complex of a size similar to LHC and a high molecular mass complex. A homolog of Alb3.1, Alb3.2, is present in Chlamydomonas, with 37% sequence identity and 57% sequence similarity. Based on the phenotype of ac29, these two genes appear to have mostly nonredundant functions.