Periplastidal Compartment Research Articles

Mapping of subcellular local pH in the marine diatom Phaeodactylum tricornutum.

Diatoms are one of the most important phytoplankton on Earth. They comprise at least ten thousand species and contribute to up to 20% of the global primary production. Because of serial endosymbiotic events and horizontal gene transfers, diatoms have developed a "secondary plastid" bounded by four membranes containing a large phase-separated compartment, termed the pyrenoid. However, the physiological significance of this unique chloroplast morphology is poorly understood. Characterization of fundamental physiological parameters such as local pH in various subcellular compartments should facilitate a greater understanding of the physiological roles of the unique structure of the secondary plastid. A promising method to estimate local pH is the in situ expression of the pH-sensitive green fluorescent protein. Here, we first developed the molecular tool for the mapping of in situ local pH in the diatom Phaeodactylum tricornutum by heterologously expressing pHluorin2 in the cytosol, periplastidal compartment (PPC; the space in between two sets of outer and inner chloroplast envelopes), chloroplast stroma, and the pyrenoid matrix. Our data suggested that PPC and the pyrenoid matrix are more acidic than the adjacent areas, the cytosol and the chloroplast stroma. Finally, absolute pH values at each compartment were estimated from the ratiometric fluorescence of a recombinant pHluorin2 protein, giving pH values of approximately 7.9, 6.8, 8.0, and 7.5 respectively, for the cytosol, PPC, stroma, and pyrenoid of the P. tricornutum cells, indicating the occurrence of pH gradients and the associated electrochemical potentials at their boundary.

Nuclear genome sequence of the plastid-lacking cryptomonad Goniomonas avonlea provides insights into the evolution of secondary plastids

BackgroundThe evolution of photosynthesis has been a major driver in eukaryotic diversification. Eukaryotes have acquired plastids (chloroplasts) either directly via the engulfment and integration of a photosynthetic cyanobacterium (primary endosymbiosis) or indirectly by engulfing a photosynthetic eukaryote (secondary or tertiary endosymbiosis). The timing and frequency of secondary endosymbiosis during eukaryotic evolution is currently unclear but may be resolved in part by studying cryptomonads, a group of single-celled eukaryotes comprised of both photosynthetic and non-photosynthetic species. While cryptomonads such as Guillardia theta harbor a red algal-derived plastid of secondary endosymbiotic origin, members of the sister group Goniomonadea lack plastids. Here, we present the genome of Goniomonas avonlea—the first for any goniomonad—to address whether Goniomonadea are ancestrally non-photosynthetic or whether they lost a plastid secondarily.ResultsWe sequenced the nuclear and mitochondrial genomes of Goniomonas avonlea and carried out a comparative analysis of Go. avonlea, Gu. theta, and other cryptomonads. The Go. avonlea genome assembly is ~ 92 Mbp in size, with 33,470 predicted protein-coding genes. Interestingly, some metabolic pathways (e.g., fatty acid biosynthesis) predicted to occur in the plastid and periplastidal compartment of Gu. theta appear to operate in the cytoplasm of Go. avonlea, suggesting that metabolic redundancies were generated during the course of secondary plastid integration. Other cytosolic pathways found in Go. avonlea are not found in Gu. theta, suggesting secondary loss in Gu. theta and other plastid-bearing cryptomonads. Phylogenetic analyses revealed no evidence for algal endosymbiont-derived genes in the Go. avonlea genome. Phylogenomic analyses point to a specific relationship between Cryptista (to which cryptomonads belong) and Archaeplastida.ConclusionWe found no convincing genomic or phylogenomic evidence that Go. avonlea evolved from a secondary red algal plastid-bearing ancestor, consistent with goniomonads being ancestrally non-photosynthetic eukaryotes. The Go. avonlea genome sheds light on the physiology of heterotrophic cryptomonads and serves as an important reference point for studying the metabolic “rewiring” that took place during secondary plastid integration in the ancestor of modern-day Cryptophyceae.

Iron-Sulfur Cluster Biosynthesis in Algae with Complex Plastids.

Plastids surrounded by four membranes harbor a special compartment between the outer and inner plastid membrane pair, the so-called periplastidal compartment (PPC). This cellular structure is usually presumed to be the reduced cytoplasm of a eukaryotic phototrophic endosymbiont, which was integrated into a host cell and streamlined into a plastid with a complex membrane structure. Up to date, no mitochondrion or mitochondrion-related organelle has been identified in the PPC of any representative. However, two prominent groups, the cryptophytes and the chlorarachniophytes, still harbor a reduced cell nucleus of symbiont origin, the nucleomorph, in their PPCs. Generally, many cytoplasmic and nucleus-located eukaryotic proteins need an iron–sulfur cofactor for their functionality. Beside some exceptions, their synthesis is depending on a so-called iron–sulfur complex (ISC) assembly machinery located in the mitochondrion. This machinery provides the cytoplasm with a still unknown sulfur component, which is then converted into iron–sulfur clusters via a cytosolic iron–sulfur protein assembly (CIA) machinery. Here, we investigated if a CIA machinery is present in mitochondrion-lacking PPCs. By using bioinformatic screens and in vivo-localizations of candidate proteins, we show that the presence of a PPC-specific CIA machinery correlates with the presence of a nucleomorph. Phylogenetic analyses of PPC- and host specific CIA components additionally indicate a complex evolution of the CIA machineries in organisms having plastids surrounded by four membranes.

Viridiuvalis adhaerens gen. et sp. nov., a novel colony-forming chlorarachniophyte.

A new chlorarachniophyte, Viridiuvalis adhaerens gen. et sp. nov. was isolated from the mucus on a coral reef from Zanpa Beach, Okinawa, Japan. The main vegetative stage of V. adhaerens consisted of unicellular coccoid cells with cell walls, although sarcinoid colonies and uniflagellate zoospores were also observed. V. adhaerens had chloroplasts with nucleomorphs and pyrenoids that were completely embedded in the chloroplast. A deep plate-like invagination of the periplastidal compartment (PPC) almost partitioned the pyrenoid and chloroplast components, which were surrounded by two membranes. The nucleomorph was positioned in the base of the invagination of the PPC. Molecular phylogenetic analyses using rRNA genes showed that V. adhaerens branched as a sister lineage of the Amorphochlora clade. The sarcinoid colony, pyrenoid embedded in the chloroplast, and nucleomorph located at the base of the deep invagination of the PPC have not been reported in other chlorarachniophytes. Based on these morphological and ultrastructural characteristics and the results of the molecular phylogenetic analyses, we propose V. adhaerens as a new genus and species of chlorarachniophyte.

In vivo Localization Studies in the Stramenopile Alga Nannochloropsis oceanica

The tiny eustigmatophyte Nannochloropsis sp. recently emerged as a promising model organism for biotechnology as it possesses a considerably high cellular oil content interesting for biodiesel production. Furthermore, the alga was shown to be genetically well accessible providing powerful tools for biotechnological engineering as well as basic research. Nannochloropsis sp. can be transformed very efficiently taking advantage of homologous recombination, however, so far it remained unclear whether these organisms are also suitable model systems for in vivo protein localization studies due to their small cell size. Here we present, to our knowledge, the first protein localization studies based on the expression of chimeric fluorescent fusion proteins in the genus Nannochloropsis using N. oceanica CCMP1779 as a model organism. Besides expressing a cytosolic green fluorescent protein (GFP), the reporter could be directed into subcellular structures such as the mitochondria, the endoplasmic reticulum and secretory pathway as well as the complex plastid including the periplastidal compartment and the stroma via fusion of specific N-terminal targeting sequences. These results expand the potential of N. oceanica as a model system in biotechnology as well as cellular biology for which now an almost complete molecular tool set exists.

Polyploidy of Endosymbiotically Derived Genomes in Complex Algae

Chlorarachniophyte and cryptophyte algae have complex plastids that were acquired by the uptake of a green or red algal endosymbiont via secondary endosymbiosis. The plastid is surrounded by four membranes, and a relict nucleus, called the nucleomorph, remains in the periplastidal compartment that is the remnant cytoplasm of the endosymbiont. Thus, these two algae possess four different genomes in a cell: Nuclear, nucleomorph, plastid, and mitochondrial. Recently, sequencing of the nuclear genomes of the chlorarachniophyte Bigelowiella natans and the cryptophyte Guillardia theta has been completed, and all four genomes have been made available. However, the copy number of each genome has never been investigated. It is important to know the actual DNA content of each genome, especially the highly reduced nucleomorph genome, for studies on genome evolution. In this study, we calculated genomic copy numbers in B. natans and G. theta using a real-time quantitative polymerase chain reaction approach. The nuclear genomes were haploid in both species, whereas the nucleomorph genomes were estimated to be diploid and tetraploid, respectively. Mitochondria and plastids contained a large copy number of genomic DNA in each cell. In the secondary endosymbioses of chlorarachniophytes and cryptophytes, the endosymbiont nuclear genomes were highly reduced in size and in the number of coding genes, whereas the chromosomal copy number was increased, as in bacterial endosymbiont genomes. This suggests that polyploidization is a general characteristic of highly reduced genomes in broad prokaryotic and eukaryotic endosymbionts.

Overexpression of Molecular Chaperone Genes in Nucleomorph Genomes

Chlorarachniophytes and cryptophytes possess complex plastids that were acquired by the ingestion of a green and red algal endosymbiont, respectively. The plastids are surrounded by four membranes, and a relict nucleus, called the nucleomorph, remains in the periplastidal compartment, which corresponds to the remnant cytoplasm of the endosymbiont. Nucleomorphs contain a greatly reduced genome that possesses only several hundred genes with high evolutionary rates. We examined the relative transcription levels of the genes of all proteins encoded by the nucleomorph genomes of two chlorarachniophytes and three cryptophytes using an RNA-seq transcriptomic approach. The genes of two heat shock proteins, Hsp70 and Hsp90, were highly expressed under normal conditions. It has been shown that molecular chaperone overexpression allows an accumulation of genetic mutations in bacteria. Our results suggest that overexpression of heat shock proteins in nucleomorph genomes may play a role in buffering the mutational destabilization of proteins, which might allow the high evolutionary rates of nucleomorph-encoded proteins.

Localization of putative carbonic anhydrases in the marine diatom, Thalassiosira pseudonana

Thirteen putative carbonic anhydrase (CA) genes have been identified in the marine multipolar centric diatom, Thalassiosira pseudonana, and two of these CAs have been localized previously. The first, an alpha CA (TpαCA1), was localized in the chloroplast stroma; the second, a zeta-type CA (TpζCA1), was localized to the periplasmic space. In the present study, cloning and localization of the remaining CAs were carried out. TpγCA2, TpγCA3, TpγCA4, TpγCA5, TpδCA1, TpδCA2, TpδCA3, and TpζCA1 were responsive to CO2 availability at the transcriptional level, being significantly reduced in cells grown at 0.4% CO2, whereas TpαCA1, TpαCA2, TpαCA3, TpγCA1, and TpδCA4 transcript levels were constitutive with respect to CO2 concentration. Full-length cDNAs for TpγCA1, TpγCA2, TpγCA3, TpγCA4, TpδCA1, and TpδCA2 were isolated and fused with the enhanced-green fluorescent gene at their 3' termini. These GFP-fusion constructs were transformed into T. pseudonana, and the resulting GFP fusion products were localized using fluorescence microscopy. The δ-type TpδCA1 was localized on the periphery of the cell, strongly suggesting localization to the periplasmic space or the frustule. The δ-type TpδCA3 and the γ-type TpγCA2 were, respectively, localized in a periplastidal compartment and the cytosol. The δ-type TpδCA2, and the γ-types TpγCA1, 3, and 4 were localized in the mitochondria. The distribution of CAs in T. pseudonana contrasts notably with that of the raphid pennate diatom P. tricornutum, with likely consequences for CCM function including modes of CO2 acquisition, regions in which DIC is accumulated, and needs for minimizing CO2 leakage from the chloroplast.

Localization of enzymes relating to C4 organic acid metabolisms in the marine diatom, Thalassiosira pseudonana

In the genome of the marine diatom-Thalassiosira pseudonana, there are several putative genes encoding enzymes potentially constitute a classical C4 type biochemical CO2-concentrating mechanism. Two genes encode a carboxylation enzyme phosphoenolpyruvate carboxylase (PEPC)1 and PEPC2; and another two encode decarboxylation enzymes, NAD(+)-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). These genes were tagged by the enhanced-green fluorescence protein, egfp, ligated in the transformation vector, and transformed into the cells of T. pseudonana for localization of GFP fusion products. The PEPC1:GFP fusion was localized at the matrix of the periplastidal compartment, while the PEPC2:GFP fusion was localized at the mitochondria. The NAD-ME:GFP fusion was localized in the cytosol and the PEPCK:GFP fusion at the mitochondria. The transcripts level of NAD-ME was extremely low, and PEPCK transcript was significantly induced under the dark, suggesting that PEPCK is involved in the dark metabolism such as respiration and amino acid metabolism in the mitochondria. Treatments of low-CO2grown T. pseudonana cells with inhibitors for PEPCK and PEPC efficiently dissipated the maximum rate of photosynthesis while these treatments did not affect high-affinity photosynthesis. These data strongly suggest that classical C4 enzymes play little role in the CCM in T. pseudonana.

Distribution of the SELMA Translocon in Secondary Plastids of Red Algal Origin and Predicted Uncoupling of Ubiquitin-Dependent Translocation from Degradation

Protein import into complex plastids of red algal origin is a multistep process including translocons of different evolutionary origins. The symbiont-derived ERAD-like machinery (SELMA), shown to be of red algal origin, is proposed to be the transport system for preprotein import across the periplastidal membrane of heterokontophytes, haptophytes, cryptophytes, and apicomplexans. In contrast to the canonical endoplasmic reticulum-associated degradation (ERAD) system, SELMA translocation is suggested to be uncoupled from proteasomal degradation. We investigated the distribution of known and newly identified SELMA components in organisms with complex plastids of red algal origin by intensive data mining, thereby defining a set of core components present in all examined organisms. These include putative pore-forming components, a ubiquitylation machinery, as well as a Cdc48 complex. Furthermore, the set of known 20S proteasomal components in the periplastidal compartment (PPC) of diatoms was expanded. These newly identified putative SELMA components, as well as proteasomal subunits, were in vivo localized as PPC proteins in the diatom Phaeodactylum tricornutum. The presented data allow us to speculate about the specific features of SELMA translocation in contrast to the canonical ERAD system, especially the uncoupling of translocation from degradation.

Dual targeting of aminoacyl-tRNA synthetases to the mitochondrion and complex plastid in chlorarachniophytes

In plants, many nucleus-encoded proteins are targeted to both mitochondria and plastids, and this process is generally mediated by ambiguous N-terminal targeting sequences that are recognized by receptors on both organelles. In many algae, however, plastids were acquired by secondarily engulfing green or red algae, which were retained within the endomembrane system. Protein targeting to these secondary plastids is more complex, and because they do not reside directly in the cytoplasm, dual targeting cannot function as it does in plant cells. Here we investigate dual targeting of aminoacyl-tRNA synthetases (aaRSs) in chlorarachniophytes, which are complex algae that possess secondary plastids and a relict nucleus derived from a green algal endosymbiont. Chlorarachniophytes have four genome-containing compartments, but almost all the aaRSs are nucleus-encoded and present in fewer than four copies (some as few as two), suggesting multiple targeting. We characterized the subcellular localization of two classes, HisRS (three copies) and GlyRS (two copies), using GFP fusion proteins. In both cases, one copy was dually targeted to mitochondria and plastids, but unlike plants this was mediated by translation initiation variants. We also found that the periplastidal compartment (the relict green algal cytoplasm) lacks both GlyRS and a cognate tRNA, suggesting that pre-charged host tRNAs are imported into this compartment. Leader analysis of other aaRSs suggests that alternative translation is a common strategy for dual targeting in these complex cells. Overall, dual targeting to mitochondria and plastids is a shared feature of plastid-bearing organisms, but the increased complexity of trafficking into secondary plastids requires a different strategy.

Nucleus‐ and nucleomorph‐targeted histone proteins in a chlorarachniophyte alga

The plastid of chlorarachniophytes is distinguished by the retention of a relict nucleus (nucleomorph) derived from a green algal endosymbiont, which is located in the periplastidal compartment (PPC). The nucleomorph genome of a chlorarachniophyte, Bigelowiella natans, encodes several plastid-targeted proteins and hundreds of housekeeping proteins, but it lacks many fundamental genes to maintain itself. Here we report the first two host nucleus-encoded genes for proteins targeted to the nucleomorph, histone H2A and H2B. We identified 20 histone genes from the host nuclear genome, and based on phylogenetic analyses predicted that most of these are derived from the host, but that two histone genes are symbiont-derived. The genes both encode N-terminal extensions resembling PPC targeting signals, further suggesting they function in the nucleomorph. Using green fluorescent protein (GFP) fusion proteins expressed in transformed cells, we confirmed that the putative symbiont H2A and H2B were targeted into the nucleomorph, whereas putative host proteins were localized to the host nucleus. Furthermore, we have developed a method to temporarily synchronize B. natans cells, and confirmed that both host and symbiont histone expression is controlled during the cell cycle. Our findings provide the first evidence of how the nucleomorph may be regulated by host-encoded gene products.

In Silico and In Vivo Investigations of Proteins of a Minimized Eukaryotic Cytoplasm

Algae with secondary plastids such as diatoms maintain two different eukaryotic cytoplasms. One of them, the so-called periplastidal compartment (PPC), is the naturally minimized cytoplasm of a eukaryotic endosymbiont. In order to investigate the protein composition of the PPC of diatoms, we applied knowledge of the targeting signals of PPC-directed proteins in searches of the genome data for proteins acting in the PPC and proved their in vivo localization via expressing green fluorescent protein (GFP) fusions. Our investigation increased the knowledge of the protein content of the PPC approximately 3-fold and thereby indicated that this narrow compartment was functionally reduced to some important cellular functions with nearly no housekeeping biochemical pathways.

New mechanistic insights into pre-protein transport across the second outermost plastid membrane of diatoms

Chromalveolates like the diatom Phaeodactylum tricornutum arose through the uptake of a red alga by a phagotrophic protist, a process termed secondary endosymbiosis. In consequence, the plastids are surrounded by two additional membranes compared with primary plastids. This plastid morphology poses additional barriers for plastid-destined proteins, which are mostly nucleus-encoded. Recent investigations have focused on the postulated translocon of the second outermost membrane (periplastidal membrane, PPM). These studies identified a symbiont-specific ERAD (endoplasmic reticulum-associated degradation)-like machinery (SELMA), which has been implicated in plastid pre-protein import. Despite this recent progress, key factors for protein transport via SELMA are still unknown. As SELMA substrates presumably undergo ubiquitination, a corresponding ubiquitin ligase and an enzyme for the subsequent removal of ubiquitin need to reside in the space between the second and third membrane (periplastidal compartment, PPC). Here we characterize two proteins fulfilling these criteria. We show that ptE3P (P.t ricornutumE3 enzyme of the PPC), the ubiquitin ligase, and ptDUP (P.t ricornutumde-ubiquitinating enzyme of the PPC), the de-ubiquitinase, localize to the PPM and PPC, respectively. In addition, we demonstrate their retained functionality by in vitro data.

Characterization of Periplastidal Compartment–Targeting Signals in Chlorarachniophytes

Secondary plastids are acquired by the engulfment and retention of eukaryotic algae, which results in an additional surrounding membrane or pair of membranes relative to the more familiar primary plastids of land plants. In most cases, the endocytosed alga loses its eukaryotic genome as it becomes integrated, but in two algal groups, the cryptophytes and chlorarachniophytes, the secondary plastids retain a vestigial nucleus in the periplastidal compartment (PPC), the remnant eukaryotic cytoplasm between the inner and the outer membrane pairs. Many essential housekeeping genes are missing from these reduced genomes, suggesting that they are now encoded in the host nucleus and their products are targeted to the PPC. One such nucleus-encoded, PPC-targeted protein, the translation elongation factor like (EFL) was recently identified in chlorarachniophytes. It bears an N-terminal-targeting sequence comprising a signal peptide and a transit peptide-like sequence (TPL) similar to the plastid-targeted proteins of chlorarachniophytes as well as a hydrophilic C-terminal extension rich in lysine and aspartic acid. Here, we characterize the function of the N- and C-terminal extensions of PPC-targeted EFL in transformed chlorarachniophyte cells. Using green fluorescent protein as a reporter molecule, we demonstrate that several negatively charged amino acids within the TPL are essential for accurate targeting to the PPC. Our findings further reveal that the C-terminal extension functions as a PPC retention signal in combination with an N-terminal plastid-targeting peptide, which suggests that plastid and PPC proteins may be sorted in the PPC.

ERAD-Derived Preprotein Transport across the Second Outermost Plastid Membrane of Diatoms

The diatom Phaeodactylum tricornutum harbors a plastid that is surrounded by four membranes and evolved by way of secondary endosymbiosis. Like land plants, most of its plastid proteins are encoded as preproteins on the nuclear genome of the host cell and are resultantly redirected into the organelle. Because two more membranes are present in diatoms than the one pair surrounding primary plastids, the targeting situation is obviously different and more complex. In this work, we focus on preprotein transport across the second outermost plastid membrane -- an issue that was experimentally inaccessible until now. We provide first indications that our hypothesis of an ERAD (ER-associated degradation)-derived preprotein transport system might be correct. Our data demonstrate that the symbiont-specific Der1 proteins, sDer1-1 and sDer1-2, form an oligomeric complex within the second outermost membrane of the complex plastid. Moreover, we present first evidence that the complex interacts with transit peptides of preproteins being transported across this membrane into the periplastidal compartment but not with transit peptides of stromal-targeted proteins. Thus, the sDer1 complex might have an additional role in discriminating preproteins that are transported across the two outermost membranes from preproteins directed across all four membranes of the complex plastid. Altogether, our studies of the symbiont-specific ERAD-like machinery of diatoms suggest that a preexisting cellular machinery was recycled to fulfill a novel function during the transition of a former free-living eukaryote into a secondary endosymbiont.

Partenskyella glossopodia gen. et sp. nov., the First Report of a Chlorarachniophyte that Lacks a Pyrenoid

A new chlorarachniophyte, Partenskyella glossopodia gen. et sp. nov., is described from a culture isolated from the Mediterranean Sea pelagic waters and maintained as strain RCC365 at the Roscoff Culture Collection (France). Vegetative cells of P. glossopodia are non-motile naked spherical cells. However, flagellate and amoeboid stages are also present in its life cycle. The cells are 2-4mum in diameter containing a pale-green, cup-shaped chloroplast, 1-2 mitochondria, a nucleus, and a Golgi apparatus. Vesicles containing storage product-like material are also present. The chloroplast is surrounded by four membranes possessing a nucleomorph in the periplastidal compartment. The minute cell size and the absence of a pyrenoid at any stage of the life cycle are unique characteristics among the chlorarachniophytes, which justifies our proposition for a new genus for strain RCC365.

Protein Targeting into the Complex Plastid of Cryptophytes

The cryptophyte Guillardia theta harbors a plastid surrounded by four membranes. This turns protein targeting of nucleus-encoded endosymbiont localized proteins into quite a challenge, as the respective precursors have to pass either all four membranes to reach the plastid stroma or only the outermost two membranes to enter the periplastidal compartment. Therefore two sets of nuclear-encoded proteins imported into the endosymbiont can be distinguished and their topogenic signals may serve as good indicators for studying protein targeting and subsequent transport across the outermost membranes of the cryptophyte plastid. We isolated genes encoding enzymes involved in two different biochemical pathways, both of which are predicted to be localized inside the periplastidal compartment, and compared their topogenic signals to those of precursor proteins for the plastid stroma, which are encoded on either the nucleus or the nucleomorph. By this and exemplary in vitro and in vivo analyses of the topogenic signal of one protein localized in the periplastidal compartment, we present new data implicating the mechanism of targeting and transport of proteins to and across the outermost plastid membranes. Furthermore, we demonstrate that one single, but conserved amino acid is the triggering key for the discrimination between nucleus-encoded plastid and periplastidal proteins.

The smallest known eukaryotic genomes encode a protein gene: towards an understanding of nucleomorph functions.

Cryptomonads are unicellular algae with plastids surrounded by four membranes. Between the two pairs of membranes lies a periplastidal compartment that harbours a DNA-containing organelle, termed the nucleomorph. The nucleomorph is the vestigial nucleus of a phototrophic, eukaryotic endosymbiont. Subcloning of parts of one nucleomorph chromosome revealed a gene coding for an Hsp70 protein. We demonstrate the expression of this nucleomorph protein-coding gene and present a model for protein transport from the host to the endosymbiont compartment.

The cryptomonad histone H4-encoding gene: structure and chromosomal localization.

Cryptomonads are unicellular flagellates whose plastids are surrounded by four membranes. A periplastidal compartment, containing eukaryote-type ribosomes, starch grains and a so-called nucleomorph, is located between the inner and outer membrane pairs. The nucleomorph has been shown to be the vestigial nucleus of a eukaryotic endosymbiont. In order to obtain more information about the chromatin structure of the nucleomorph and the host nuclear chromosomes, we studied the distribution of the histone, H4. H4 was not detectable in the nucleomorph by immunolocalization, thus supporting earlier findings by Gibbs [In: Wiesner et al. (Eds.), Experimental Phycology 1, 1990, pp. 145-157]. Likewise, no H4 DNA was demonstrable in the nucleomorph by Southern hybridization. Sequence analysis, and Southern and Northern blot data of a cryptomonad gene, H4, indicate an intermediate position for these genes between animals and plants.