CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

Aquaporin Nt-TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes.

Members of the major intrinsic protein (MIP) family, described in plants as water-selective channels (aquaporins), can also transport small neutral solutes in other organisms. In the present work, we characterize the permeability of plant vacuolar membrane (tonoplast; TP) and plasma membrane (PM) to non-electrolytes and evaluate the contribution of MIP homologues to such transport. PM and TP vesicles were purified from tobacco suspension cells by free-flow electrophoresis, and membrane permeabilities for a wide range of neutral solutes including urea, polyols of different molecular size, and amino acids were investigated by stopped-flow spectrofluorimetry. For all solutes tested, TP vesicles were found to be more permeable than their PM counterparts, with for instance urea permeabilities from influx experiments of 74.9 +/- 9.6 x 10(-6) and 1.0 +/- 0.3 x 10(-6) cm sec-1, respectively. Glycerol and urea transport in TP vesicles exhibited features of a facilitated diffusion process. This and the high channel-mediated permeability of the same TP vesicles to water suggested a common role for MIP proteins in water and solute transport. A cDNA encoding a novel tonoplast intrinsic protein (TIP) homologue named Nicotiana tabacum TIPa (Nt-TIPa) was isolated from tobacco cells. Immunodetection of Nt-TIPa in purified membrane fractions confirmed that the protein is localized in the TP. Functional expression of Nt-TIPa in Xenopus oocytes showed this protein to be permeable to water and solutes such as urea and glycerol. These features could account for the transport selectivity profile determined in purified TP vesicles. These results support the idea that plant aquaporins have a dual function in water and solute transport. Because Nt-TIPa diverges in sequence from solute permeable aquaporins characterized in other organisms, its identification also provides a novel tool for investigating the molecular determinants of aquaporin transport selectivity.

Purified vesicles of tobacco cell vacuolar and plasma membranes exhibit dramatically different water permeability and water channel activity.

The vacuolar membrane or tonoplast (TP) and the plasma membrane (PM) of tobacco suspension cells were purified by free-flow electrophoresis (FFE) and aqueous two-phase partitioning, with enrichment factors from a crude microsomal fraction of >/=4- to 5-fold and reduced contamination by other cellular membranes. For each purified fraction, the mean apparent diameter of membrane vesicles was determined by freeze-fracture electron microscopy, and the osmotic shrinking kinetics of the vesicles were characterized by stopped-flow light scattering. Osmotic water permeability coefficients (Pf) of 6.1 +/- 0.2 and 7.6 +/- 0.9 microm . s(-1) were deduced for PM-enriched vesicles purified by FFE and phase partitioning, respectively. The associated activation energies (Ea; 13.7 +/- 1.0 and 13.4 +/- 1.4 kcal . mol(-1), respectively) suggest that water transport in the purified PM occurs mostly by diffusion across the lipid matrix. In contrast, water transport in TP vesicles purified by FFE was characterized by (i) a 100-fold higher Pf of 690 +/- 35 microm . s(-1), (ii) a reduced Ea of 2.5 +/- 1.3 kcal . mol(-1), and (iii) a reversible inhibition by mercuric chloride, up to 83% at 1 mM. These results provide functional evidence for channel-mediated water transport in the TP, and more generally in a higher plant membrane. A high TP Pf suggests a role for the vacuole in buffering osmotic fluctuations occurring in the cytoplasm. Thus, the differential water permeabilities and water channel activities observed in the tobacco TP and PM point to an original osmoregulatory function for water channels in relation to the typical compartmentation of plant cells.

Identification of calreticulin-like protein as one of the phosphoproteins modulated in response to oligogalacturonides in tobacco cells.

Oligogalacturonide-induced modifications of protein phosphorylation in cells of Nicotiana tabacum L. were investigated by in-vitro phosphorylation of plasma-membrane-enriched fractions and electrophoretic analysis on two-dimensional gels. About 100 polypeptides were resolved; among these 40 phosphoproteins were detected and their 33P-labelling quantified. Most of the phosphorylations were inhibited by staurosporine and several proteins were hyperphosphorylated in the presence of okadaic acid, indicating the presence of protein phosphatase(s) in addition to staurosporine-susceptible protein kinase(s) in the plasma-membrane-enriched fraction. In the presence of oligogalacturonides, phosphorylation of seven acidic polypeptides ranging from 15 to 65 kDa was strongly enhanced. A twofold enhancement of the phosphorylation of 24-kDa protein and a two- to threefold decrease in the phosphorylation of acidic proteins of MrS 62, 65, 80 and 84 was also observed in response to oligogalacturonides. One of the oligogalacturonide-modulated phosphoproteins was identified as calreticulin by direct nucleotide sequencing after preparative two-dimensional electrophoresis and comparison with protein database sequences. Decreased phosphorylation of calreticulin was also observed in vivo, shortly after addition of oligogalacturonides to tobacco cells, confirming the biological relevance of the modification. Although the presence of calreticulin, an abundant reticuloplasmin with high calcium-binding capacity, has been reported in both mammalian and plant cells, its function is as yet largely unknown. Modulation of the phosphorylation of a plant calreticulin-like protein by oligogalacturonides is shown here, suggesting a role in the early transduction steps of this signal.

Erythrocyte behavior in free-flow electrophoresis is independent of erythrocyte age.

Enzyme activities (acetylcholinesterase, fluorescein diacetate hydrolase, superoxide dismutase), membrane protein band 4.1a/4.1b ratio and density of various fractions of rabbit erythrocytes separated did not show differences between various fractions of erythrocytes separated by free-flow electrophoresis. Also, electric field deflection of lightest and densest fractions of erythrocytes did not differ. These results point to the lack of changes in surface charge density during in vivo erythrocyte aging.