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Mitochondrial FtsZ in a Chromophyte Alga

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Science  18 Feb 2000:
Vol. 287, Issue 5456, pp. 1276-1279
DOI: 10.1126/science.287.5456.1276

Abstract

A homolog of the bacterial cell division gene ftsZ was isolated from the alga Mallomonas splendens. The nuclear-encoded protein (MsFtsZ-mt) was closely related to FtsZs of the α-proteobacteria, possessed a mitochondrial targeting signal, and localized in a pattern consistent with a role in mitochondrial division. Although FtsZs are known to act in the division of chloroplasts, MsFtsZ-mt appears to be a mitochondrial FtsZ and may represent a mitochondrial division protein.

Mitochondria are ubiquitous organelles that form networks, reticulae, or punctate structures in eukaryotic cells. Mitochondria in many cells appear to constitutively fuse with one another and divide (1), but we know little about the proteins involved in these processes, particularly mitochondrial division. Eukaryotes depend on mitochondria for respiration and adenosine triphosphate synthesis and rely on them to divide before daughter mitochondria can be apportioned to each new cell generation. In chloroplasts, homologs of the bacterial cell division protein FtsZ are essential components of the organellar division machinery (2). FtsZ is found in nearly all prokaryotes, is structurally related to tubulin, and accumulates at the furrow between dividing cells, playing a critical role in cell division (3). No potential mitochondrial FtsZ has been identified in the complete genomes of Caenorhabditis elegans orSaccharomyces cerevisiae. However, because both mitochondria and chloroplasts arose from endosymbiotic bacteria, we anticipated that early in evolution, mitochondrial division might also have been regulated by FtsZ. Here we describe two distinct complementary DNAs for FtsZ homologs from an algal flagellate: One appears to encode an FtsZ of the chloroplast, and the second appears to encode an FtsZ of the mitochondrion.

We screened a cDNA library of the unicellular chromophyte (stramenopile) alga Mallomonas splendens(4) with two probes: (i) part of the chloroplastAtFtsZ1-1 gene of the plant Arabidopsis thaliana(5) and (ii) a fragment of the ftsZ1 gene of the α-proteobacterium Sinorhizobium meliloti(6). The screens recovered two cDNAs, and Southern blot analyses confirmed that each corresponded to a different, single-copy, nuclear gene in M. splendens. TheArabidopsis probe detected a Mallomonas ftsZhomolog that we call MsFtsZ-cp(M allomonas splendens FtsZchloroplast; GenBank accession numberAF120117), because sequence analysis showed a translation product with clear affinities to other known chloroplast FtsZ proteins. MsFtsZ-cp was unequivocally allied to chloroplast FtsZs from the red algaeCyanidium and Galdieria, the cryptomonad algaGuillardia, the plants Arabidopsis and pea, and the moss Physcomitrella, which in turn are related to FtsZs of cyanobacteria, from which chloroplasts derive (Fig. 1). We thus believe that MsFtsZ-cp has a role in dividing theMallomonas chloroplast.

Figure 1

Phylogeny of FtsZ showing the position of the two Mallomonas proteins. Major bacterial groups are represented as triangles, with the number of taxa therein. TheMallomonas chloroplast FtsZ (MsFtsZ-cp) is allied with other algal chloroplast FtsZs (Galdieria, Cyanidium, and Guillardia) with very high levels of confidence. TheMallomonas mitochondrial FtsZ (MsFtsZ-mt) is unequivocally the sister to α-proteobacteria, the group from which mitochondria derive. This is a quartet puzzling tree for 52 sequences with 306 sites. Numbers at the nodes refer to percentage bootstrap support in distance, frequency in 1000 quartet puzzling replicates, and percentage bootstrap support in parsimony (Paup 4.0b2a). Dashes at the nodes of the tree indicate less than 50% support. Alignment and accession numbers are available on request.

The second screen, probing for sequences related to the α-proteobacterial gene, identified a second Mallomonas ftsZ homolog, which we call MsFtsZ-mt(M allomonas splendens FtsZmitochondrion; GenBank accession numberAF120116). Phylogenetic analyses (Fig. 1) of the predicted 401–amino acid protein (relative molecular mass 42310) demonstrated that MsFtsZ-mt was most closely related to FtsZs of α-proteobacteria—the nearest known bacterial relatives of mitochondria (7). Northern blot analysis revealed aMsFtsZ-mt transcript of the expected size (∼1.25 kb) in mRNA isolated from asynchronous cultures of M. splendens. MsFtsZ-mt has an NH2-terminal extension predicted to act as a basic, amphipathic, mitochondrial targeting sequence (8).

The mitochondria of M. splendens are, like those of most other cells, highly plastic and dynamic (1,9). At any one time, they number 15 to 30 per cell and can rapidly (over several seconds) change shape between being spherical, ellipsoid, elongate, or platelike. They move within the cell and undergo frequent fission and fusion events (10). Viewed within living cells at interphase, at least one mitochondrion (usually several) is medially constricted (Fig. 2B shows examples in fixed cells), and these dividing mitochondria become completely separated within 1 min (10). Observations of the fusion of mitochondria (10) suggest that the tip of one mitochondrion meets and fuses with the end or side of a second mitochondrion, similar to mitochrondrial fusion, in other eukaryotes (1, 9).

Figure 2

Anti–MsFtsZ-mt labels protein associated with the mitochondria of M. splendens. (A) Western blot analysis of total cellular protein from M. splendensprobed with anti–MsFtsZ-mt and detected with alkaline phosphatase–conjugated secondary antibodies is shown. The single immunoreactive band of ∼42 kD is the expected size of the MsFtsZ-mt protein. (B through I) Confocal immunofluorescence microscopy of M. splendens(17, 18) labeled with MitoTracker CMXRos [red channel (B)] and anti–MsFtsZ-mt [green channel (C)]; colocalization of mitochondria and anti–MsFtsZ-mt appears in yellow in the overlays of the two channels [(D) through (I)]. (B) through (D) show an entire cell. Three mitochondria that are medially constricted and possibly dividing are indicated by asterisks in (B). In (D), arrowheads and arrows indicate examples of medial and peripheral (respectively) anti–MsFtsZ-mt labeling of mitochondria. (E) through (I) are consecutive sections through part of a cell showing three mitochondria, each with a distinct medial belt of MsFtsZ-mt (arrowheads). Scale bars, 4 μm in (B) and 2 μm in (E).

To determine the location of MsFtsZ-mt in M. splendens, we generated antisera to bacterially expressed protein (11)(Fig. 2A). Confocal microscopy of immunolabeled interphase cells (Fig. 2, B through I) showed that MsFtsZ-mt was always associated with mitochondria and displayed patches of labeling in two distinct locations: around the middle of mitochondria (medial localization) and at the edges of mitochondria (peripheral localization). Medial localization was often associated with mitochondria that were constricted (arrowheads, Fig. 2D) and possibly represented MsFtsZ-mt in dividing organelles, similar to the localization of FtsZ in dividing bacteria (3). A series of confocal sections showed three mitochondria, each with a medial belt of anti–MsFtsZ-mt labeling (Fig. 2, E through I); although these mitochondria were not obviously constricted, they may have been in the very early stages of division. In bacteria, the FtsZ ring forms before there are any morphological indicators (such as cell shape) of division (12). Peripheral localization of MsFtsZ-mt (arrows, Fig. 2D) appeared as one to several patches per mitochondrion and often occurred at mitochondrial poles. The similarities between the localization of MsFtsZ-mt in M. splendens and the behavior of bacterial FtsZ suggest that MsFtsZ-mt acts in mitochondrial division. Furthermore, the distribution of MsFtsZ-mt is strikingly similar to that shown by Dnm1, a dynamin-related guanosine triphosphatase that regulates mitochondrial division in yeast and C. elegans (13). Dnm1 is found at the constricted regions of apparently dividing mitochondria (similar to the medial localization of MsFtsZ-mt) and at the ends of mitochondrial tubules that may have recently completed division (similar to some of the peripheral localizations of MsFtsZ-mt). However, Dnm1 and other dynamin-like proteins are located on the mitochondrial outer membrane (13, 14), whereas MsFtsZ-mt has no predicted membrane-spanning domain and is likely to be transported to the inside of the mitochondrion.

To assess the submitochondrial localization of MsFtsZ-mt experimentally, the protein (including the putative mitochondrial targeting sequence) was expressed in yeast as a green fluorescent protein (GFP) fusion (15). Mitochondria purified from the transgenic yeast carried a protein corresponding in size to the MsFtsZ-mt fusion, and immunoblotting revealed MsFtsZ-mt within the mitochondrial compartment, not displayed on its surface (Fig. 3A). Confocal fluorescence microscopy showed that MsFtsZ-mt localized to yeast mitochondria (Fig. 3B), which exist as a reticulum around the cell cortex. The transgenic yeast appeared to have slightly abnormal mitochondrial morphologies: The mitochondria of most transformed cells displayed one or more bright nodes of fluorescence (Fig. 3, B, D, and E; of 100 cells, 68 had one node, 23 had two to four nodes, and 9 had no nodes of fluorescence), which were never observed in untransformed cells or in cells transformed with GFP targeted by fusion to other mitochondrial proteins.

Figure 3

MsFtsZ-mt cDNA tagged with GFP is targeted to the yeast mitochondrion. (A) Mitochondria were purified from yeast cells transformed with MsFtsZ-mt/GFP, subjected to limited proteolysis with trypsin, and then analysed by SDS–polyacrylamide gel electrophoresis and immunoblotting (15). With an antibody to GFP, MsFtsZ-mt/GFP (FtsZ) was detected in mitochondria (lane 1) and in mitochondria treated with trypsin (lane 2), but was degraded by trypsin when the membranes were ruptured by osmotic shock (lane 3). Control immunoblots reveal the fate of the surface-located protein Tom70 and the intermembrane space protein cyt b2. (B through F) Confocal fluorescence microscopy (17) of yeast cells transformed with the MsFtsZ-mt/GFP fusion. (B) and (D) show green fluorescence from the MsFtsZ-mt/GFP fusion; arrows indicate bright nodes of fluorescence in the mitochondria. (C) is the corresponding phase contrast image of (B). (E) shows co-staining of the cells in (D) with the mitochondrial-specific dye Mitotracker CMXRos (Molecular Probes) as described (9). (F) is the overlay of (D) and (E) revealing (in yellow) colocalization of the MsFtsZ-mt/GFP fusion with mitochondria. Scale bars, 2 μm.

If mitochondrial FtsZs are, in fact, absent from the genomes of all fungi and animals, then it is possible that the dynamin-like proteins have taken over the role of FtsZ in mitochondrial division in these organisms. However, MsFtsZ-mt seems to be able to affect mitochondrial morphology even in an organism such as yeast that normally relies on Dnm1 for organelle division. It seems likely that there will be major mechanistic differences in mitochondrial fission catalyzed by dynamins or FtsZs: one protein working from the outside of the organelle and the other from the inside.

We conclude that MsFtsZ-mt is likely to have been acquired from an endosymbiotic α-proteobacterium that was the ancestor of the present-day mitochondrion. We suggest that in the course of evolution, the gene was transferred from mitochondrion to nucleus (16) and that the nuclear-encoded protein is now targeted back to the mitochondrion to play a role in the division of the organelle. This is the first identification of a eukaryoticftsZ whose protein seems to be specifically targeted to the mitochondrion, and which may thus be related to the earliest mitochondrial division genes. The Mallomonas mitochondrial FtsZ will be a useful key for identifying other components critical to moulding the shape and facilitating the intergenerational transmission of mitochondria.

Note added in proof: A possible mitochondrial FtsZ from a red alga has recently been reported. The predicted protein ofCmftsZ1 (GenBank accession number AB032071) is 53% identical to MsFtsZ-mt and clusters with MsFtsZ-mt and the proteobacteria in a phylogenetic analysis like that in Fig. 1.

  • * To whom correspondence should be addressed. E-mail: plbeech{at}deakin.edu.au

  • Present address: Ludwig Institute for Cancer Research, Royal Melbourne Hospital, 3050, Australia.

  • Present address: The Scripps Research Institute, 11055 North Torrey Pines Road, La Jolla, CA 92037, USA.

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