Gene Transfer from Organelles to the Nucleus: How Much, What Happens, and Why?1

Abstract

Chloroplasts were once free-living cyanobacteria, mitochondria were once free-living proteobacteria, and both have preserved remnants of eubacterial genomes. But from the functional standpoint, both organelles have retained much more of their eubacterial biochemistry than is reflected in their DNA. The discrepancy between the number of genes that organelles encode and the number of eubacterial proteins that they contain is generally explained by something that we have come to know as “endosymbiotic gene transfer.” During evolution, organelles export their genes to the nucleus, but reimport the products with the help of transit peptides and protein-import machinery, so that proteins are retained in organelles, but most of the genes are not. This process, over time, concentrates genetic material in nuclear chromosomes. Because gene-regulatory processes under the control of the nucleus are more complex and interrelated than those under the control of organelles, and because organelles naturally tend to come under the control of nuclear regulatory genes (imagine the opposite!), organelle regulatory processes are likely to have been among the first to be transferred successfully to the nucleus. From the standpoint of genes, this process therefore results in a compartmented, but integrated, eukaryotic genetic system under the regulatory dominance of the nucleus (Herrmann, 1997), rather than genetically semiautonomous organelles. However, from the standpoint of the encoded products of transferred genes, a surprising picture is emerging that could be loosely described as “a funny thing happened on the way back to the organelle.” The prerequisite for endosymbiotic gene transfer is protein-import machinery in the two membranes that surround chloroplasts and mitochondria, which allows these organelles to take up cytosolic precursors, cleave the transit peptides, and release the processed polypeptides into the stroma and matrix, respectively. For an overview of what proteins that machinery consists of, how it works, and how it might have evolved, we recommend the recent overviews by Schatz and Dobberstein (1996) for a general summary, and Heins et al. (1998) for chloroplasts in particular. The first clear-cut examples of endosymbiotic gene transfer became known about 10 years ago (for review, see Gray [1992] for a general overview; Brennicke et al. [1993] for the process of gene transfer). Here we provide a brief summary of organelle genome reduction and its impact on plant cells, skimming the surface with a few examples of gene transfer to the nucleus from both plastids and mitochondria. From the standpoint of gene product function, we will consider factors that (a) might influence the immediate fate of genes that become transferred to the nucleus, and (b) might help to determine whether such transfer events become genetically fixed. We will also consider the question of why genes tend to be transferred from organelles to the nucleus. In 1998 we have an unfair advantage relative to those who wondered about the genetic “semiautonomy” of chloroplasts in 1978, because we have a much better overview of the number and types of genes contained in plastid genomes. Several chloroplast genomes have been completely sequenced, quite a few more are now being sequenced, and a cyanobacterial genome has been sequenced, with additional ones in the pipeline. How many proteins are encoded by ctDNA? The answer depends upon which plastid one considers; a summary is given in TableI (see also Martin et al., 1998). The nonphotosynthetic plastids in Epifagus (a parasite of beech trees) and Plasmodium (a parasite of humans) contain about 20 protein-coding genes. Fully functional higher plant chloroplasts encode about 60 to 80 proteins, the rhodophyte Porphyra can boast 200, whereas Odontella and Cyanophoraencode on the order of 120 to 130 proteins. By contrast, the genome of the unicellular cyanobacterium Synechocystis encodes about 3168 proteins. Table I reveals that plastid genomes generally encode many more proteins than even the largest mitochondrial genomes studied (Lang et al., 1997; Unseld et al., 1997), but on the whole, they contain only about 1% to 5% as many protein-coding genes as a comparatively small cyanobacterial genome. Size comparison of sequenced chloroplast genomes, three large mitochondrial genomes, and a cyanobacterial genome Due to length limitations in this forum, accession numbers (in parentheses) rather than references are given for plastids. Lang et al. (1997). Oda et al. (1992). Unseld et al. (1997). Kaneko et al. (1996). Size comparison of sequenced chloroplast genomes, three large mitochondrial genomes, and a cyanobacterial genome Due to length limitations in this forum, accession numbers (in parentheses) rather than references are given for plastids. Lang et al. (1997). Oda et al. (1992). Unseld et al. (1997). Kaneko et al. (1996). How many proteins do plastids contain? In an earlier work, Ellis (1981)suggested that the roughly 200 chloroplast proteins then directly resolvable by two-dimensional electrophoresis could be just the “tip of the iceberg.” More recently it was shown that thylakoid membranes alone contain at least 75 major proteins (Herrmann et al., 1991; Pillen et al., 1996). We can roughly estimate the number of plastid proteins, considering the number of identified genes in theSynechocystis genome that belong to pathways and the functions known to reside partially, predominantly, or exclusively in plastids. Only about 50% (approximately 1600) of the genes in theSynechocystis genome have a known or putative function (Kaneko et al., 1996). Using the table provided by Kaneko et al. (1996), a rough calculation reveals that of those 1600 genes inSynechocystis, homologs of about 600 might be expected to exist in plastids. This provides a rough lower boundary for the number of chloroplast proteins. Assuming that the other approximately 1500 genes of as-yet-unassignable function in Synechocystisharbor another 400 to 500 probable plastid functions, one can obtain a conservative estimate of about 1000 different proteins that might be contained in a fully functional plastid. However, this estimate assumes that plastids do not do more for the plant cell than cyanobacteria do for themselves, which is probably not true (they probably do more). We estimate that the total number of different proteins, including, for example, isoenzymes and proteins involved in plastid-nucleus gene regulatory circuitry, in various types of plastids may be closer to about 2000. However, estimations from Arabidopsis data suggest that this number could approach 5000 (R. Douce, personal communication). Thus, plastids import the vast majority of their proteins, which are encoded in the nucleus. How many plant nuclear genes are known that descend from cyanobacterial genomes? To our knowledge, nobody has yet compared all 3168Synechocystis proteins to the plant databases and recorded/reported the results in such a manner that would answer that question. We do know, however, that at least 44 genes found in at least one plastid genome have functional, structurally characterized homologs in the nucleus of at least one higher plant (Martin et al., 1998). Surprisingly, genes tend to undergo multiple parallel losses from ctDNA in independent evolutionary lineages; parallel losses even outnumber phylogenetically unique losses by a ratio of about 4 to 1 (Martin et al., 1998). Protein-coding genes found in plastidial, mitochondrial, and a cyanobacterial genome in the functional categories used by Kaneko et al. (1996) (see alsohttp://www.kazusa.or.jp/cyano/cyano.html). For organelles, the sum of all genes found in the genomes listed in Table I was used, whereby a gene found in 10 genomes is counted as 1 gene, not 10. Obviously, the largest plastidial (Porphyra) and mitochondrial (Reclinomonas) genomes contribute the majority of different genes per category. Localization of several enzymes of central carbohydrate metabolism in spinach. Suggested evolutionary origins for the nuclear genes are color coded. Enzymes regulated through the thioredoxin system are indicated. Many enzymes in the figure are allosterically regulated, but no allosteric regulation is indicated here. Enyzme abbreviations are: FBA, Fru-1,6-bisphosphate aldolase; FBP, Fru-1,6-bisphosphatase; GAPDH, glyceraldehyde-3-P dehydrogenase; PGK, 3-phosphoglycerate kinase; PRI, Rib-5-P isomerase; PRK, phosphoribulokinase; RPE, ribulose-5-P 3-epimerase; SBP, sedoheptulose-1,7-bisphosphatase; TKL, transketolase; TPI, triosephosphate isomerase; TAL, transaldolase; GPI, Glc-6-P isomerase; G6PDH, Glc-6-P dehydrogenase; 6GPDH, 6-phosphogluconate dehydrogenase; pGluM, phosphoglucomutase; PGM, phosphoglyceromutase; PFK, phosphofructokinase (pyrophosphate and ATP-dependent); ENO, enolase; PYK, pyrtuvate kinase; PDC, pyruvate dehydrogenase complex (E1, E2, E3 components); and T, translocator. PDC is a multienzyme complex, but only one set of components is drawn here. Note that chloroplast isoenzymes of PGM, ENO, and PYK have not been demonstrated in spinach leaves, but for convenience we have included those enzymes in this figure, since they have been well characterized in the plastids of other higher plants (Plaxton, 1996). Open arrowheads indicate transport rather than conversion. Solid arrowheads indicate physiologically irrerversible reactions. For details, see Martin and Schnarrenberger (1997), Johnston et al. (1997; A and B subunits of pyruvate dehydrogenase), Nowitzki et al. (1998; chloroplast and cytosolic GPI), and Wenderoth et al. (1997; chloroplast and cytosolic G6PDH). It seems likely that the nuclear genes for chloroplast and cytosolic FBA (C. Schnarrenberger, personal communication) and G6PDH (A. von Schaewen, personal communication) are of mitochondrial origin, but archaebacterial sequences are still not known for comparison, so the color coding for these enzymes is dark. See also Fischer et al. (1997) and Lange et al. (1998). First, all proteins shown in the figure are encoded in the nucleus except the large subunit of Rubisco, which is plastid encoded. In several algae, chloroplast phosphoglycerate mutase and the subunits of chloroplast pyruvate dehydrogenase are also plastid encoded (Martin et al., 1998). Second, several enzymes in chloroplasts are not cyanobacterial proteins, but rather are proteobacterial proteins encoded by genes of mitochondrial origin (Martin and Schnarrenberger, 1997) and acquired in the nucleus the targeting signals (transit peptide), which redirect them to the chloroplast, where they replaced the function of the preexisting cyanobacterial homolog. Obviously, this is most likely to occur for proteins that were common to the eubacterial antecedents of both mitochondria and chloroplasts, and thus are functionally redundant in eukaryotes through endosymbiosis (Martin and Schnarrenberger, 1997). Third, higher plants possess a largely eubacterial glycolytic pathway in the cytosol. This is an unexpected finding, because the host of mitochondrial origins was, from the standpoint of today's views, either a descendant of the archaebacteria or possibly an archaebacterium outright (Doolittle, 1998). Archaebacteria possess the enzymes of the glycolytic and gluconeogenetic pathways, but for the enzymes shown in the figure, the ancestry of the “host” is not reflected as archaebacterial enzymes in the cytosol. Rather, the archaebacterial enzymes have been replaced by the products of genes that were donated to the nucleus from eubacterial symbionts, chloroplasts, and mitochondria. These genes have not acquired a region encoding a transit peptide (which is simpler than acquiring one) and therefore their products have been left “stranded” in the cytosol (Fig. 2). There are still quite a few gray areas in the figure where either the higher plant sequences have not been determined or they are known but the gene phylogeny is insufficiently clear (in our view) to make a statement on the origin of the plant nuclear genes. Chloroplastic and cytosolic pyruvate kinases are a good example of sequenced genes with an evolutionary history that is so intriguingly complex (Hattori et al., 1995) that one cannot yet tell where the plant nuclear genes come from. Furthermore, cases are also known in which the compartmentation of individual gene products can change in different lineages over evolutionary time, such that Figure 2, if prepared forChlamydomonas or Euglena rather than spinach, would reveal different patterns of origins and compartmentation for the enzymes of the same pathways in those organisms (for an overview, see Martin and Schnarrenberger, 1997). Schematic diagram contrasting the fate of products of transferred genes under two simple scenarios (see text). Prom., Promoter and other regulatory elements necessary for gene expression; Tr.Pep., transit peptide. Gray arrows symbolize processes in time. The genetic systems of plastids and the nucleus are quite different. Genome and gene organization in plastids is generally, but not purely, prokaryotic. Transcription occurs with the help of both a plastid-encoded prokaryotic-type RNA polymerase and a nuclear-encoded, single-chain, phage-like, 110-kD RNA polymerase (scRPO) (Hedtke et al., 1997), and different sets of genes are specifically transcribed by these two types of polymerases in plastids (Hajdukiewicz et al., 1997; Kapoor et al., 1997). So when a plastid gene finds its way to the nucleus, by whatever means, it moves from a genetic apparatus that is compact, operon harboring, and intron poor, to one that is inflated, operon splitting, and intron laden (Herrmann, 1997). Such a gene has an immediate problem. It must become selected to avoid becoming a pseudogene (“promiscuous see 1997), and its product must become can from the of the gene product is to with its many plastid-encoded for to in the its gene must elements and targeting Obviously, targeting signals are of of transferred genes in the nucleus in a more to endosymbiotic gene transfer than the of the signals necessary for the gene product to its This is by the that of a transit peptide is not as as one might 1 in sequences from successfully the import of proteins into mitochondria in and By contrast, of into plant occurs at a much lower et al., in rather than of targeting might be for the of chloroplast genes into nuclear over time. Thus, to all plastids with product in to are for of the may be more to than that genes that are transferred to the nucleus from plastids or mitochondria may undergo a in which the gene but a transit peptide. In that the encoded product would be a cytosolic the gene the the that cytosolic of gene which descend from plastids or mitochondria, such as higher plant cytosolic phosphoglycerate (Martin and Schnarrenberger, 1997), might a in the this the first of endosymbiotic gene transfer would and nuclear of a transferred gene to the of a in The product so would about the possibly with preexisting cytosolic functions, in the of functionally redundant with We consider simple for such transferred genes, upon the of their products with cytosolic proteins. the donated gene product the of the cell better than the preexisting cytosolic as has been for the origin of the eubacterial glycolytic pathway in the eukaryotic cytosol (for a see Martin and Schnarrenberger, 1997; Martin and Nowitzki et al., then it can be expected that the gene for the cytosolic would be and the of would in way be to the of examples of such cytosolic are in It would only be a of events et al., or et al., to a with which could with the in the organelle to of the the preexisting and products are functionally then it a of as to which one For transfer of could be expected a results in a in the nucleus encoding a the function of the product is more to the of the cell than the preexisting cytosolic or if it has no preexisting with which to then it will be from and will In this it is a of the gene (a) a (b) into something that may be for the or signals to the product into the from which the gene into a different (see recent example in which transferred genes may have to encode something different can be found in the chloroplast import machinery and 1998). the product with cytosolic functions in a manner that is to the nuclear will be The of would in a manner to the of the transferred gene, and this has been by communication) as a that might why genes tend to in organelles, rather than be transferred to the nucleus (Herrmann, 1997). one of the events in eukaryotic cell is the export of a into the a where the not belong et al., that gene products can be when in the of the that genes, which to the proteins, have a of in which the encoded product was from or in which were in the product was to to the This can be expected to in a of more of in such genes, and thus in of in nuclear of organelle genes relative to their examples known where nuclear genes of organelle origin up transit peptides from preexisting nuclear et al. (1996) a clear-cut genes for a mitochondrial There are two genes for in the nuclear genome that and a pseudogene for in the mitochondrial genome. nuclear encode transit peptides, and in a we can see by where the transit peptides come from. In the of the transit peptide was from the transit peptide in the nuclear gene for mitochondrial a of the of the transit peptide is found on two different nuclear and In the the region encoding the mitochondrial subunit has of its transit peptide from of the transit peptide in the nuclear gene for mitochondrial subunit These events the of and in the of transit peptides et al., 1996). are also known where a in the of transit A clear was by et al. (1996), who found that the transit peptide for mitochondrial in was acquired by between the nuclear gene for and a gene for a cytosolic glyceraldehyde-3-P The transit peptide of the nuclear gene was from the first three of a gene for a glycolytic functionally a peptide that used to be of a into a mitochondrial targeting et al., 1996). can have an additional on the fate of transferred genes, since can directly influence gene at various and 1997). Thus, whether by in coding sequences or in these examples indicate that transit peptides are not to once a gene is in the nucleus and genes tend to be transferred from chloroplasts to the nucleus in the first Several factors that might the transfer of genes to the nucleus were in by and (1996), who the of functions in organelles that might the for genes in organelles, thus their transfer to the nucleus. This is one other factors it be that complex gene regulation is only in the because plastid gene is regulated, and in a complex manner (Herrmann, 1997), both at the (Hajdukiewicz et al., 1997) and and 1997) regulation in plastids is not a it is of an eukaryotic gene regulation system (Herrmann, 1997). This is for example, in the that the genes for factors by the plastid-encoded RNA polymerase are and regulated by the nuclear apparatus et al., 1997), as is the gene for the RNA polymerase of plastids (Hedtke et al., 1997). So gene regulation in plastids is to the nucleus, but that not directly why genes tend to rather than reside in plastids they can be regulated as not what were once free-living the first plastid its host it became genetically from its free-living it probably became to when it is of It cannot the that are to in its genome. This is known as time, is to or to How figure into gene transfer from organelles to the a gene is successfully transferred to the nucleus, it moves from a to a and the gene from the fate of In the this therefore the transfer of genes to the nucleus, a to for the of in organelle genomes? In has been shown to a of in genes of mitochondria 1996). Furthermore, that have as for many of of genetically in the of also clear of in their genomes, as of in various genes relative to their free-living 1996). In plant mitochondria and chloroplasts, however, the is more In these organelles, the of is much than in mitochondria 1997). the of in plant organelles is not higher than in the nucleus, as would it is lower et al., This that factors are at in plant organelles, which the of The most of these is genetic between organelles, as is well known in chloroplasts of et al., 1996). might also be provided by the of chloroplasts, which between genomes the same plastid such that on ctDNA could be do import a of cyanobacterial that is functionally involved in and in chloroplasts, that these pathways may be in and plastids et al., pathways that might help to for the lower of such as have not yet been characterized from plastids. But one gene involved in this pathway is encoded in the mitochondrial genome of an where it might influence the mitochondrial et al., in plant organelles may have an influence on their lower and by the of would be for to take Obviously, alone not in for the transfer of genes from organelles to the nucleus, but in the of organelle when the majority of organelle genome is to have it may have a we were to 500 years and then Table I for the same organelle genomes, we would probably numbers of genes and in cases the number would possibly we do not to that because in eukaryotic organelles, the genome has been in by the nuclear genome. are organelles of they descend from the same as mitochondria, but no are known that possess a genome (Martin and 1998). In the process of endosymbiotic gene transfer has to From mitochondrial genomes we can obtain an of what types of genes are the to be from these those for and (Fig. In no so is left to proteins of and the can be as well (Herrmann, 1997). if we were to back the a years or we would be to genes are the first to be from This is a more but from the standpoint of today's data it that genes for regulatory functions tend to be in the nucleus more than or functions (for an overview, see 1997). of this are in several chloroplast proteins (in to the factors for the plastid-encoded RNA polymerase The of the chloroplast a regulatory for the The subunits and of the are encoded in all plant chloroplast genomes sequenced to but the regulatory is nuclear encoded in all plants studied to and is therefore probably the first gene of this complex to have been transferred (Herrmann, 1997). The same in can be found the chloroplast the subunit is ctDNA encoded in all higher plants whereas the regulatory subunit is nuclear encoded (Martin et al., 1998). Rubisco, a in of plastid-encoded and regulation can be These are possibly of a general for regulation and to be in the nucleus. In many factors figure into endosymbiotic gene time, gene the cell into the nuclear But once a gene has been the nuclear the from which the gene product can in a functionally gene product to an the organelle gene can be Here we have a few factors that could help to why the of organelle genome reduction in evolution, which could also help to the but of genes to the nucleus We von Schaewen, and Schnarrenberger for