The vacuolar membrane or tonoplast (TP) and the plasma membrane (PM) of tobacco suspension cells were purified by free-f low 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-f low light scattering. Osmotic water permeability coefficients (P f ) of 6.1 ؎ 0.2 and 7.6 ؎ 0.9 m⅐s ؊1 were deduced for PM-enriched vesicles purified by FFE and phase partitioning, respectively. The associated activation energies (E a ; 13.7 ؎ 1.0 and 13.4 ؎ 1.4 kcal⅐mol ؊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 P f of 690 ؎ 35 m⅐s ؊1 , (ii) a reduced E a of 2.5 ؎ 1.3 kcal⅐mol ؊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 P f suggests a role for the vacuole in buffering osmotic f luctuations 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.In plants, the cell wall continuum and the cell-to-cell communications (plasmodesmata) provide privileged paths for water exchange and equilibration. Cellular membranes also critically control water transport in a variety of functions, including cell volume and turgor regulation and long-distance transport in nonvascular tissues (1). These functions involve the ability of membranes to transport or sequester osmotic solutes as well as their intrinsic permeability to water (1, 2). The presence in most plant cells of a large vacuole suggests that intra-and transcellular water exchange may depend on the permeability of two membranes in series, the plasma membrane (PM) and the vacuolar membrane or tonoplast (TP) (3, 4). Pressure probe measurements have brought about a better understanding of water relations in a variety of higher plant cell types (1, 5). However, this technique only provides access to the overall cell hydraulic conductivity that includes the water permeabilities of the PM and the TP plus those of the cell wall and the plasmodesmata. The individual water permeabilities of the two membranes thus remain to be determined in these cells (6).The possible existence of water-transporting pores or water channels in membranes of higher plants was discussed nearly 40 years ago (7,8). However, experimental approaches to the molecular mechanisms of membrane water permeability in these organisms h...

By its ability to engage in a variety of redox reactions and coordinating metals, cysteine serves as a key residue in mediating enzymatic catalysis, protein oxidative folding and trafficking, and redox signaling. The thiol redox system, which consists of the glutathione and thioredoxin pathways, uses the cysteine residue to catalyze thiol-disulfide exchange reactions, thereby controlling the redox state of cytoplasmic cysteine residues and regulating the biological functions it subserves. Here, we consider the thiol redox systems of Escherichia coli and Saccharomyces cerevisiae, emphasizing the role of genetic approaches in the understanding of the cellular functions of these systems. We show that although prokaryotic and eukaryotic systems have a similar architecture, they profoundly differ in their overall cellular functions.

A facilitated diffusion for glycerol is present in human erythrocytes. Glycerol transporters identified to date belong to the Major Intrinsic Protein (MIP) family of integral membrane proteins, and one of them, aquaporin-3 (AQP3), has been characterized in mammals. Using an antibody raised against a peptide corresponding to the rat AQP3 carboxyl terminus, we examined the presence of AQP3 in normal and Colton-null (aquaporin-1 (AQP1)-deficient) human erythrocytes. Three immunoreactive bands were detected on immunoblots of both normal and Colton-null red cells, very similar to the bands revealed in rat kidney, a material in which AQP3 has been extensively studied. By immunofluorescence, anti-AQP3 antibodies stained the plasma membranes of both normal and Colton-null erythrocytes. Glycerol transport was measured on intact erythrocytes by stopped-flow light scattering and on one-step pink ghosts by a rapid filtration technique. Glycerol permeability values, similar in both cell types, suggest that AQP1 does not represent the major path for glycerol movement across red blood cell membranes. Furthermore, pharmacological studies showed that Colton-null red cells remain sensitive to water and glycerol flux inhibitors, supporting the idea that another proteinaceous path, probably AQP3, mediates most of the glycerol movements across red cell membranes and represents part of the residual water transport activity found in AQP1-deficient red cells.Human erythrocytes are highly permeable to water, urea, and glycerol (1-3). The existence of membrane proteins that facilitate water and solute movements in these cells has been postulated, but most of these proteins remain to be characterized. In 1992, Agre and co-workers identified a very abundant protein of the human red blood cell as the first water-selective channel that was named aquaporin-1 (AQP1) 1 (4). Colton-null erythrocytes, lacking AQP1, exhibit a reduced osmotic water permeability (5). However, these cells are found to be residually permeable to water, suggesting the presence of additional, non-AQP1, water channels.The existence of a protein-mediated glycerol transport in erythrocytes has relied mostly on pharmacological evidence. In particular, inhibition of glycerol transport by sulfhydryl reagents, phloretin, and copper ions has been reported (1,3,6). Yet, the identity of the erythrocyte glycerol carrier remains unknown. The glycerol facilitators identified to date all belong to the Major Intrinsic Protein (MIP) family (7). They have been characterized in several organisms: GlpF, a bacterial glycerol permease facilitator (8); Fps1p, a yeast glycerol exporter (9); aquaporin-3 (AQP3), initially characterized in mammalian kidney (10 -12); and AQP7, recently identified in rat testis (13). Compared with other mammalian aquaporins, which are selective mostly for water, AQP3 is moderately permeable to water, but highly permeable to glycerol and possibly to urea (11,12,14). AQP3 expression has been reported in several mammalian tissues, including kidney, intestine, stomach...

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