Colocalization of plastid division proteins in the chloroplast stromal compartment establishes a new functional relationship between FtsZ1 and FtsZ2 in higher plants.

Chloroplast division is driven by a macromolecular complex containing components that are positioned on the cytosolic surface of the outer envelope, the stromal surface of the inner envelope, and in the intermembrane space. The only constituents of the division apparatus identified thus far are the tubulin-like proteins FtsZ1 and FtsZ2, which colocalize to rings at the plastid division site. However, the precise positioning of these rings relative to the envelope membranes and to each other has not been previously defined. Using newly isolated cDNAs with open reading frames longer than those reported previously, we demonstrate here that both FtsZ2 proteins in Arabidopsis, like FtsZ1 proteins, contain cleavable transit peptides that target them across the outer envelope membrane. To determine their topological arrangement, protease protection experiments designed to distinguish between stromal and intermembrane space localization were performed on both in vitro imported and endogenous forms of FtsZ1 and FtsZ2. Both proteins were shown to reside in the stromal compartment of the chloroplast, indicating that the FtsZ1- and FtsZ2-containing rings have similar topologies and may physically interact. Consistent with this hypothesis, double immunofluorescence labeling of various plastid division mutants revealed precise colocalization of FtsZ1 and FtsZ2, even when their levels and assembly patterns were perturbed. Overexpression of FtsZ2 in transgenic Arabidopsis inhibited plastid division in a dose-dependent manner, suggesting that the stoichiometry between FtsZ1 and FtsZ2 is an important aspect of their function. These studies raise new questions concerning the functional and evolutionary significance of two distinct but colocalized forms of FtsZ in plants and establish a revised framework within which to understand the molecular architecture of the plastid division apparatus in higher plants.

FtsZ ring formation at the chloroplast division site in plants.

Among the events that accompanied the evolution of chloroplasts from their endosymbiotic ancestors was the host cell recruitment of the prokaryotic cell division protein FtsZ to function in chloroplast division. FtsZ, a structural homologue of tubulin, mediates cell division in bacteria by assembling into a ring at the midcell division site. In higher plants, two nuclear-encoded forms of FtsZ, FtsZ1 and FtsZ2, play essential and functionally distinct roles in chloroplast division, but whether this involves ring formation at the division site has not been determined previously. Using immunofluorescence microscopy and expression of green fluorescent protein fusion proteins in Arabidopsis thaliana, we demonstrate here that FtsZ1 and FtsZ2 localize to coaligned rings at the chloroplast midpoint. Antibodies specific for recognition of FtsZ1 or FtsZ2 proteins in Arabidopsis also recognize related polypeptides and detect midplastid rings in pea and tobacco, suggesting that midplastid ring formation by FtsZ1 and FtsZ2 is universal among flowering plants. Perturbation in the level of either protein in transgenic plants is accompanied by plastid division defects and assembly of FtsZ1 and FtsZ2 into filaments and filament networks not observed in wild-type, suggesting that previously described FtsZ-containing cytoskeletal-like networks in chloroplasts may be artifacts of FtsZ overexpression.

Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis.

In higher plants, two nuclear gene families, FtsZ1 and FtsZ2, encode homologs of the bacterial protein FtsZ, a key component of the prokaryotic cell division machinery. We previously demonstrated that members of both gene families are essential for plastid division, but are functionally distinct. To further explore differences between FtsZ1 and FtsZ2 proteins we investigated the phenotypes of transgenic plants overexpressing AtFtsZ1-1 or AtFtsZ2-1, Arabidopsis members of the FtsZ1 and FtsZ2 families, respectively. Increasing the level of AtFtsZ1-1 protein as little as 3-fold inhibited chloroplast division. Plants with the most severe plastid division defects had 13- to 26-fold increases in AtFtsZ1-1 levels over wild type, and some of these also exhibited a novel chloroplast morphology. Quantitative immunoblotting revealed a correlation between the degree of plastid division inhibition and the extent to which the AtFtsZ1-1 protein level was elevated. In contrast, expression of an AtFtsZ2-1 sense transgene had no obvious effect on plastid division or morphology, though AtFtsZ2-1 protein levels were elevated only slightly over wild-type levels. This may indicate that AtFtsZ2-1 accumulation is more tightly regulated than that of AtFtsZ1-1. Plants expressing the AtFtsZ2-1 transgene did accumulate a form of the protein smaller than those detected in wild-type plants. AtFtsZ2-1 levels were unaffected by increased or decreased accumulation of AtFtsZ1-1 and vice versa, suggesting that the levels of these two plastid division proteins are regulated independently. Taken together, our results provide additional evidence for the functional divergence of the FtsZ1 and FtsZ2 plant gene families.

Enzymology of cottonseed microsomal N-acylphosphatidylethanolamine synthase: kinetic properties and mechanism-based inactivation.

An ATP-, Ca2+-, and CoA-independent acyltransferase activity, designated "N-acylphosphatidylethanolamine (NAPE) synthase", was reported to catalyze the direct acylation of phosphatidylethanolamine (PE) with free fatty acids (FFAs) in cottonseed microsomes [K.D. Chapman, T.S. Moore, Jr., Plant Physiol. 102 (3) (1993) 761-769]. Here, NAPE synthase was purified 138, 176-fold from crude cottonseed homogenates to a specific activity of 5.98 mumol min-1 mg-1 protein by immobilized artificial membrane chromatography. Enzyme purity was confirmed by the presence of a 64 kDa polypeptide in fractions analyzed by tricine-SDS-PAGE. Initial velocity measurements with various free fatty acids ([14C]-linoleic, -palmitic, -oleic, -stearic and -myristic acids) and saturating concentrations of dioleoyl-PE revealed non-Michaelis-Menten, biphasic kinetics with high and low affinity sites demonstrating positive cooperativity specific for each [14C]-FFA. In contrast to FFA substrates, no kinetic differences were observed for two different molecular species of PE, (18:1,18:1)-PE and (16:0,18:2)-PE, and biphasic curves were not pronounced. Neither [14C]-dipalmitoylphosphatidylcholine nor [14C]-palmitoyl-CoA served as acyl donors for the synthesis of NAPE, indicating a preference for FFAs as the acyl donor. Also, neither ethanolamine nor sphingosine functioned as acyl acceptor molecule to form N-acylethanolamine or ceramide, respectively, indicating specificity for the phospholipid PE. NAPE synthase was inactivated in a time- and concentration-dependent manner by diisopropylfluorophosphate (DFP) through the apparent modification of one serine residue. Palmitic acid protected the enzyme from DFP-inactivation and [14C]-DFP incorporation, suggesting that a serine residue probably binds FFAs in the enzyme's active site forming an acyl-enzyme intermediate. Collectively, these results provide new information on the kinetic behavior of a purified, integral membrane enzyme which synthesizes a bilayer-stabilizing product from two lipid-soluble substrates. The biochemical properties of cottonseed NAPE synthase are consistent with a possible free fatty acid scavenging role in vivo. (c) 1998 Elsevier Science B.V.

Photoaffinity labeling of cottonseed microsomal N-acylphosphatidylethanolamine synthase protein with a substrate analogue, 12-[(4-azidosalicyl)amino]dodecanoic acid.

N-Acylphosphatidylethanolamine (NAPE), an unusual acylated derivative of phosphatidylethanolamine (PE), is synthesized from free fatty acids and PE in cotton seedlings (Chapman and Moore (1993) Plant Physiol. 102(3), 761-769). Here we use a photoreactive dodecanoic acid analogue, [12-(4-azidosalicy)amino]dodecanoic acid (ASD), and its 125I-labeled derivative to identify a protein subunit which corresponds to this cottonseed NAPE synthase activity. Dodecylmaltoside (DDM)-solubilized microsomal NAPE synthase enzyme was irreversibly and progressively inactivated by adding increasing concentrations of ASD and illuminating with UV254 light. Protection from this photoinactivation was afforded by the natural substrate, palmitic acid. In low light, microsomal NAPE synthase utilized ASD as a substrate to synthesize NAPE; palmitic acid competed for this activity. NAPE synthase activity was measured directly in gel slices following nondenaturing PAGE of DDM-solubilized microsomal membrane proteins. Two-dimensional electrophoresis (nondenaturing PAGE, followed by SDS-PAGE) of photoaffinity-labeled, DDM-solubilized microsomal proteins revealed a 64 kDa polypeptide that was associated with the active NAPE synthase enzyme. Also, a 64 kDa protein was photoaffinity labeled in all NAPE synthase isozyme fractions isolated by preparative isoelectric focusing; photoaffinity labeling of this 64 kDa polypeptide was diminished in the presence of exogenously supplied palmitic acid. Collectively, our results demonstrate that ASD specifically interacts with NAPE synthase in a manner analogous to its fatty acid substrate and indicate that a 64 kDa polypeptide is a component of cottonseed microsomal NAPE synthase. ASD will be a useful molecular probe in future studies aimed at understanding the physiological role of this NAPE synthase enzyme in membranes of plant cells.

Rapid purification of cotton seed membrane-bound N-acylphosphatidylethanolamine synthase by immobilized artificial membrane chromatography.

N-Acylphosphatidylethanolamine synthase (NAPES) is a membrane-bound enzyme present in cotton seedlings at a concentration of < or = 0.02% of the total protein. NAPES was purified to electrophoretic homogeneity in a single chromatographic step using immobilized artificial membrane (IAM) chromatography. The IAM column used for NAPES purification was etherIAM.PEC10/C3 and this surface contains a monolayer of immobilized phosphatidyl-ethanolamine (PE). Since PE is an analogue of the natural substrate for NAPES, etherIAM.PEC10/C3 columns function as an affinity column for this enzyme. Detergent-solubilized microsomal proteins from cotton were loaded on to the etherIAM.PEC10/C3 column and eluted with buffered mobile phases containing 0.2 mM dimyristoylphosphatidylethanolamine (DMPE) and 2 mM dodecylmaltoside. Little NAPES functional activity eluted if DMPE was removed from the mobile phase. Mobile phase DMPE is also a substrate for NAPES, both the mobile phase and IAM surface contains NAPES substrates. Mobile phase DMPE may function as both a surfactant-type affinity displacing ligand effecting protein elution and also a stabilizing factor of NAPES functional activity. The loading capacity on semi-preparative etherIAM.PEC10/C3 (6.5 x 1.0 cm) columns was ca. 5 mg of total detergent solubilized microsomal proteins, and protein recovery was quantitative. This one-step IAM purification of NAPES resulted in a single band on silver-stained polyacrylamide gels, and 3940 fold increase in NAPES specific activity. The molecular mass of the purified NAPES protein is 64,000. 125I labeled [12-(4-azidosalicyl)amino]dodecanoic acid is a photoreactive fatty acid substrate of NAPES that was used to confirm protein purity.