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We dissected the regulatory region of the AVP1 gene encoding the vacuolar H+-pyrophosphatase (V-PPase) of Arabidopsis thaliana by using a GUS-reporter assay system. The cloned 1.4 kb 5'-regulatory region in the GUS-reporter transgenic plants was sufficient for the light-induced repression. Furthermore, the 1.4 kb regulatory region was active in all tissues examined and its activity was especially enhanced in pollen, whereas the shorter 0.4 kb regulatory region was active only in pollen. Further detailed analyses revealed that the GUS activity in pollen was regulated by at least three cis-acting regions in an additive or synergetic manner. These findings establish a distinct mechanism of the tissue-specific regulation of V-PPase expression in developing pollen. and imply the biological significance of the V-PPase in pollen maturation.

Vacuolar H(+)-PPase, a membrane bound proton-translocating pyrophosphatase found in various species including plants, some protozoan and prokaryotes, has been demonstrated to be localized to the vacuolar membrane in plants. Using a GUS reporter system and a green fluorescent protein (GFP) fusion protein, we investigated the tissue distribution and the subcellular localization, respectively, of a novel type H(+)-PPase encoded by AVP2/AVPL1 identified in the Arabidopsis thaliana genome. We showed that AVP2/AVPL1 is highly expressed at the trichome and the filament of stamen. Furthermore, the fluorescence of GFP-tagged AVP2/AVPL1 showed small dot-like structures that were observed throughout the cytoplasm of various Arabidopsis cells under a fluorescent microscope. The distribution of this dot-like fluorescent pattern was apparently affected by a treatment with brefeldin A. Moreover, we demonstrated that most dot-like fluorescent structures colocalized with a Golgi resident protein. These findings suggest that this novel type H(+)-PPase resides on the Golgi apparatus rather than the vacuolar membrane.

DNA is flexible and easily subjected to bending and wrapping via DNA/protein interaction. DNA supercoiling is known to play an important role in a variety of cellular events, such as transcription, replication, and recombination. It is, however, not well understood how the superhelical strain is efficiently redistributed during these reactions. Here we demonstrate a novel property of an initiator protein in DNA relaxation by utilizing a one-molecule-imaging technique, atomic force microscopy, combined with biochemical procedures. A replication initiator protein, RepE54 of bacterial mini-F plasmid (2.5 kb), binds to the specific sequences (iterons) within the replication region (ori2). When RepE54 binds to the iterons of the negatively supercoiled mini-F plasmid, it induces a dynamic structural transition of the plasmid to a relaxed state. This initiator-induced relaxation is mediated neither by the introduction of a DNA strand break nor by a local melting of the DNA double strand. Furthermore, RepE54 is not wrapped by DNA repeatedly. These data indicate that a local strain imposed by initiator binding can induce a drastic shift of the DNA conformation from a supercoiled to a relaxed state.

We addressed the question of how nuclear histones and DNA interact and form a nucleosome structure by applying atomic force microscopy to an in vitro reconstituted chromatin system. The molecular images obtained by atomic force microscopy demonstrated that oligonucleosomes reconstituted with purified core histones and DNA yielded a 'beads on a string' structure with each nucleosome trapping 158 +/- 27 bp DNA. When dinucleosomes were assembled on a DNA fragment containing two tandem repeats of the positioning sequence of the Xenopus 5S RNA gene, two nucleosomes were located around each positioning sequence. The spacing of the nucleosomes fluctuated in the absence of salt and the nucleosomes were stabilized around the range of the positioning signals in the presence of 50 mM NaCl. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure.

The in vivo degradation of the plasmid-encoded tetracycline/H+ antiporter (TET) in Escherichia coli cells was studied using three mutants with carboxyl-terminal truncation at the positions in the hydrophilic carboxyl-terminal tail (TET388), in the last putative transmembrane helix XII (TET382), and immediately before the helix XII (TET365). All the mutant TET proteins were localized in the membrane. Expressed TET388 was active in transport and stable against proteolysis. However, TET382 and TET365 were inactive and proteolyzed rapidly. Thus, the importance of the helix XII for protease-resistant proper folding of TET is obvious. Interestingly, overproduced chaperonin (GroEL and GroES) partly prevented degradation of TET365.

Inorganic pyrophosphatase was purified to homogeneity from the vacuolar membranes of pumpkin hypocotyl tissue. The purified Inorganic pyrophosphatase consists of a single kind of 75-kDa polypeptide, and the stacked molecules with a repeating unit of 5.8 x 3.8 nm are seen under electron micrography. It exhibits H(+)-translocating activity across membranes coupled with PPi hydrolysis when it is reconstituted into proteoliposomes. A monovalent cation is required for the H+ translocation with the K+ ion being the most effective, but evidence for active transport of 42K+ into proteoliposomes was not obtained under the conditions tested. The hydrolysis of PPi by the reconstituted proteoliposomes is stimulated by the addition of a H+ ionophore, carbonyl cyanide p-trifluoromethyoxyphenylhydrazone, but not by a K+ ionophore, valinomycin. Both hydrolysis of PPi and PPi-dependent H+ translocation of the proteoliposomes are inhibited by N,N'-dicyclohexylcarbodiimide.

A 490 bp DNA fragment was amplified from Methanosarcina barkeri genomic DNA by the polymerase chain reaction (PCR) using oligonucleotide primers designed based on conserved amino acid sequences of the F1-ATPase beta subunits. The amino acid sequence deduced from the DNA sequence of this fragment was highly homologous to a portion of the F1-ATPase beta subunit. This indicates that this archaebacterium has a gene of F-type ATPase in addition to a gene of V-type ATPase.

Vacuolar membrane H(+)-translocating pyrophosphatase (H(+)-PPase) was purified from pumpkin seedlings. Its enzymatic properties including molecular size of constituting polypeptide (75 kDa) were very similar to those of mung bean H(+)-PPase [(1989) J. Biol. Chem. 264, 20068-20073]. The native, functional molecular size of the pumpkin H(+)-PPase was estimated to be 135-139 kDa from gel permeation HPLC of the purified enzyme in the presence of detergent and from radiation inactivation of the enzyme in vacuolar membranes. It is concluded that native, functional pumpkin H(+)-PPase, and also probably H(+)-PPases from other plants, is a dimer of 75 kDa subunits.