A characteristic of many molecular phylogenies is that the three domains of life (Bacteria, Archaea, Eucarya) are clearly separated from each other. The analyses of ancient duplicated genes suggest that the last common ancestor of all presently known life forms already had been a sophisticated cellular prokaryote. These findings are in conflict with theories that have been proposed to explain the absence of deep branching lineages. In this paper we propose an alternative scenario, namely, a large meteorite impact that wiped out almost all life forms present on the early Earth. Following this nearly complete frustation of life on Earth, two surviving extreme thermophilic species gave rise to the now existing major groups of living organisms, the Bacteria and Archaea. [The latter also contributed the major portion to the nucleo-cytoplasmic component of the Eucarya]. An exact calibration of the molecular record with regard to time is not yet possible. The emergence of Eucarya in fossil and molecular records suggests that the proposed late impact should have occurred before 2100 million years before present (BP). If the 3500 million year old microfossils [Schopf, J. W. 1993: Science 260: 640-646] are interpreted as representatives of present day existing groups of bacteria (i.e., as cyanobacteria), then the impact is dated to around 3700 million years BP. The analysis of molecular sequences suggests that the separation between the Eucarya and the two prokaryotic domains is less deep then the separation between Bacteria and Archaea. The fundamental cell biological differences between Archaea and Eucarya were obtained over a comparatively short evolutionary distance (as measured in number of substitution events in biological macromolecules). Our interpretation of the molecular record suggests that life emerged early in Earth's history even before the time of the heavy bombardment was over. Early life forms already had colonized extreme habitats which allowed at least two prokaryotic species to survive a late nearly ocean boiling impact. The distribution of ecotypes on the rooted universal tree of life should not be interpreted as evidence that life originated in extremely hot environments.

The tonoplast of isolated vacuoles from photoautotrophic suspension cells of Chenopodium rubrum L. was studied by means of the patch-clamp technique. In a symmetrical K+ concentration of 46 mM, similar to in vivo conditions, the tonoplast displayed a membrane potential near zero and a linear current-voltage relationship with a mean slope of 1.0 S/M2. ATP at 2 mM hyperpolarized the tonoplast (vacuole positive) by 15-20 mV and, in a parallel experiment, acidified the vacuole (outside pH 7.0) to pH 5.0, as monitored by accumulation of acridine orange. Analysis of the voltageclamp current indicates a 2-fold, ATP-dependent increase of the membrane capacitance, from 4 to 8 mF/m2, and an ATP-independent, unidentified ion channel having a mean opening time of about 5 msec and a conductivity of 0.5-1.0 pS. Considerable attention currently is devoted to the energetics of substrate transport across the tonoplast, the membrane separating cytoplasm and vacuole in plant cells (1). A membrane ATPase is thought to pump protons into the vacuole and thus to energize the tonoplast by a pH gradient and possibly by a membrane potential. By applying the patch-clamp technique to isolated vacuoles, we have studied the electrical properties of the tonoplast and found that the latter features an ATP-dependent electrogenic pump that creates a small membrane potential but a substantial pH gradient, revealed by parallel experiments with the pH probe acridine orange. MATERIALS AND METHODSPhotoautotrophic and phytohormone-independent suspension cells derived from hypocotyl cells of Chenopodium rubrum L. (2) were cultured as described (3). For protoplast isolation, cells from the exponential growth phase were used (for the growth pattern of the culture, see ref. 4). Cells from 6-day-old cultures were harvested from the suspension by filtration through a 15-,um-pore nylon net (Thoma, Mossingen, F.R.G.). Fifteen grams of cells (fresh weight) was suspended in 50 ml of medium I [20 mM 2-(N-morpholino)ethanesulfonic acid (Mes)/KOH, pH 5.3/0.3 M mannitol/2 mM CaCl2/10 mM KCl/1 mM DL-dithiothreitol/5 mM MgCl2/0.5% bovine serum albumin]. Fifty milliliters of medium I containing 2.5 g of cellulase TC from Trichoderma reesi (Serva, Heidelberg) and 2.5 g of pectinase 5S from Aspergillus niger (Serva) was added to the cell suspension and incubated on a gyratory shaker (120 rpm); after 90 min, there was no more Calcofluor white-staining (5) detectable at the cell surface (Calcofluor white ST solution was a gift from U. Seitz, Tubingen, F.R.G.). Protoplasts were harvested by centrifugation (100 x g, 15 min) and washed twice with medium II (20 mM Mes/KOH, pH 6/0.3 M mannitol/25 mM KCl/1 mM DL-dithiothreitol/0.5 mM MgCl2/0.1% bovine serum albumin); yield of protoplasts was 81%. For further purification, the protoplasts in medium II were loaded on a step gradient of 10, 7.5, 5, and 2.5% (wt/wt) Ficoll 400 in medium II and centrifuged (100 x g, 15 min). Spherical protoplasts (60o of the crude protoplasts) banding at the 7.5/5% (wt/wt) Ficoll 400 interfa...

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The patch clamp technique has been applied to isolated vacuoles from green suspension cells of Chenopodium rubrum to record electrical parameters of the tonoplast. In a symmetrical K+ solution of 46 mᴍ, the membrane displays a near zero voltage, whereas 2 mᴍ ATP will hyper-polarize it to 15 or 20 mV (vacuole positive). The conductance amounts to about one S · m-2. Fluctuations of the clamp current are explained by an unknown channel species having opening times of 5 - 10 ms. Together with previous work on a tonoplast vesicle preparation and unpublished data on vacuoles from our laboratory, the present results indicate an electrogenic membrane ATPase pumping protons from the cytoplasm to the vacuole.

A microsomal vesicle fraction (GV) markedly enriched by the Golgi marker enzyme latent inosine diphosphatase (IDPase) has been isolated from photoautotrophic suspension-cell protoplasts ofChenopodium rubrum L. Addition of ATP creates a substantial pH gradient across the GV membrane as measured by accumulation of acridine orange. The GV showed a density of 1.14 g·cm(-3) by equilibrium density centrifugation on sucrose gradients. Coincidence of acridine-orange accumulation and IDPase activity was confirmed on Percoll gradients. Formation of the pH gradient half-saturates at 0.3 mM MgATP, peaks at pH 7, and is competitively inhibited by ADP (k i≤0.1 mM), but not by Pi; it is hardly inhibited by orthovanadate, quickly dissipated by monensink 2=18 nM), nigericin (k 1/2=25 nM), and sluggishly by N-ethylmaleimide (k 1/2≈35 μM). Inhibition by KNO3 (k 1/2≈6.7 mM) is incomplete (60%). Uridine 5'-diphosphate (UDP)-glucose, UDP-galactose, but not UDP-mannose and the pertinent sugars, dissipate the ATP-generated pH gradient (k 1/2≈10-20 mM UDP-glucose; optimum pH at 7.8). This UDP-glucose activity is accompanied by release of Pi, but not of glucose or sucrose. UDP-glucoseinduced Pi release from the GV saturates (k 1/2=1 mM UDP-glucose; optimum pH at 7) and is completely inhibited by the anion-channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS;k 1/2=140 μM). The GV incorporates UDP-[U-(14)C]glucose into an acid-labile, alkaline-stable macromolecular compound; this process is like-wise inhibited by DIDS. We propose a model including, inter alia, a UDP-glucose/uridine-5'-monophosphate translocator and a phosphate-permeable anion channel to operate in Golgi vesicles ofChenopodium rubrum.