The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Lipid topology and physical properties of the outer mitochondrial membrane of the yeast, Saccharomyces cerevisiae

The outer membrane of yeast mitochondria was studied with respect to its lipid composition, phospholipid topology and membrane fluidity. This membrane is characterized by a high phospholipid to protein ratio (1.20). Like other yeast cellular membranes the outer mitochondrial membrane contains predominantly phosphatidylcholine (44% of total phospholipids), phosphatidylethanolamine (34%) and phosphatidylinositol (14%). Cardiolipin, the characteristic phospholipid of the inner mitochondrial membrane (13% of total phospholipids) is present in the outer membrane only to a moderate extent (5%). The ergosterol to phospholipid ratio is higher in the inner (7.0 wt%) as compared to the outer membrane (2.1 wt.%). Attempts to study phospholipid asymmetry by selective degradation of phospholipids of the outer leaflet of the outer mitochondrial membrane failed, because isolated right-side-out vesicles of this membrane became leaky upon treatment with phospholipases. Selective removal of phospholipids of the outer leaflet with the aid of phospholipid transfer proteins and chemical modification with trinitrobenzenesulfonic acid on the other hand, gave satisfactory results. Phosphatidylcholine and phosphatidylinositol are more or less evenly distributed between the two sides of the outer mitochondrial membrane, whereas the majority of phosphatidylethanolamine is oriented towards the intermembrane space. The fluidity of mitochondrial membranes was determined by measuring fluorescence anisotropy using diphenylhexatriene (DPH) as a probe. The lower anisotropy of DPH in the outer as compared to the inner membrane, which is an indication for an increased lipid mobility in the outer membrane, was attributed to the higher phospholipid to protein and the lower ergosterol to phospholipid ratio. The data presented here show, that the outer mitochondrial membrane, in spite of its close contact to the inner membrane, is distinct not only with respect to its protein pattern, but also with respect to its lipid composition and physical membrane properties.     

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris

Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.     

Separation and characterization of the outer membrane of Pseudomonas aeruginosa.

A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa [P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)] were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.     

大鼠心肌线粒体内、外膜磷脂动态结构的研究

我们以DPH为荧光探针.用毫微秒荧光分光光度计测定了大鼠心肌线粒体及线粒体内、外膜的动态微细结构;用HPLC分析了磷脂组成.实验结果提示.完整线粒体膜流动性主要反映了线粒体外膜的运动状态.线粒体内膜微粘度及磷脂分子摇动角大于外膜,扩散速率小于外膜.除去了蛋白质的线粒体内、外膜磷脂脂质体膜流动性无明显差异.提示线粒体内膜的高微粘度与膜中所含有的多量蛋白有关.     

Lipid lateral diffusion and local microviscosity in plant mitochondrial membranes with various length and unsaturation of fatty acids

Two main aspects of the lipid dynamics, local microviscosity and lateral diffusion, were investigated in intact plant mitochondria isolated from different tissues exhibiting large differences in their fatty acids in terms of unsaturation (amount of linoleic and linolenic acids) or length of the hydrocarbon chains. In addition, the same parameters were determined in the outer and inner membranes isolated from cauliflower mitochondria, which differed not only in the fatty acid composition but also by the lipid-to-protein ratio. In intact mitochondria, local microviscosity assayed with anthroyloxy-fatty acids exhibited a transverse gradient from the surface to the center of the bilayer, which was mainly affected by the unsaturation index and the content in linoleic or linolenic acids. In contrast, lipid lateral diffusion increased as the content in linolenic or palmitic acids increased, but was not directly correlated to the unsaturation index. Interestingly, local microviscosity at the membrane surface was higher in the outer membrane than in the inner membrane, whereas no significant difference was found in lipid lateral diffusion. These results indicate that the influence of the fatty acid composition of mitochondrial membranes on the dynamics of the phospholipid bilayer depends on the type of movement considered and suggest that other parameters, such as the protein content of the bilayer, also affect membrane fluidity.     

The outer membrane of Proteus mirabilis. II. The extractable lipid fraction and electron-paramagnetic resonance analysis of the outer and cytoplasmic membranes.

1. The lipid fraction extracted from the outer and cytoplasmic membranes of Proteus mirabilis with chloroform/methanol consisted almost entirely of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. 2. The phospholipid content of the cytoplasmic membrane was more than twice that of the outer membrane (38% as against 18% of the total dry weight) and the proportions of the three phospholipids differed somewhat in the two membranes. Yet, the fatty acid composition of the extractable lipids was essentially the same in both membranes. 3. The freedom of motion of spin-labeled fatty acids in the outer membrane of P. mirabilis depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe is an associate lipid structure with the properties of a bilayer. Nevertheless, the mobility of the probe was more restricted in the outer membrane than in the cytoplasmic membrane, indicating a higher viscosity of the outer membrane. 4. Chloroform/methanol completely removed the phospholipids from the outer membrane, leaving the lipopolysaccharide moiety intact. The motion of spin-labeled fatty acids in the extracted membranes was, however, highly restricted, suggesting that, in the native outer membrane, the local environment of the probe is composed of phospholipids rather than lipopolysaccharide. Aqueous acetone extraction removed only 75-80% of the phospholipids of the outer membrane. Nevertheless, the mobility of the spin-labeled fatty acid remained highly restricted, suggesting the existence of two phospholipid environments in the outer membrane differing in the nature of their association with the lipopolysaccharide and protein moieties.     

Effects of lipids on mitochondrial functions

Mitochondria contain two membranes: the outer and inner membrane. Whereas the outer membrane is particularly enriched in phospholipids, the inner membrane has an unusual high protein content and forms large invaginations termed cristae. The proper phospholipid composition of the membranes is crucial for mitochondrial functions. Phospholipids affect activity, biogenesis and stability of protein complexes including protein translocases and respiratory chain supercomplexes. Negatively charged phospholipids such as cardiolipin are important for the architecture of the membranes and recruit soluble factors to the membranes to support mitochondrial dynamics. Thus, phospholipids not only form the hydrophobic core of biological membranes that surround mitochondria, but also create a specific environment to promote functions of various protein machineries. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

Cholesterol heterogeneity in bovine rod outer segment disk membranes

Rod outer segment disk membranes have been used to study visual transduction events. Numerous studies have also focused on protein-lipid interactions in these membranes. The possible heterogeneity of the disk membrane composition has not been addressed in such studies. Freeze fracture studies (Andrews, L. D., and Cohn, A. I. (1979) J. Cell Biol. 81, 215-220; Caldwell, R., and McLaughlin, B. (1985) J. Comp. Neurol. 236, 523-537) suggest a difference in cholesterol content between newly formed and old disks. This potential heterogeneity in disk membrane composition was investigated using digitonin. Osmotically intact bovine rod outer segment disk membranes prepared by Ficoll flotation were separated based on the cholesterol content of the disks. The addition of digitonin to disk membrane suspensions in a one-to-one molar ratio with respect to cholesterol produced an increase in the density of the membranes in proportion to the amount of cholesterol present. The digitonin-treated disks were separated into subpopulations using a sucrose density gradient. Disks were shown to vary in cholesterol to phospholipid ratio from 0.30 to 0.05. The ratio of phospholipid to protein remained constant in all disk subpopulations at approximately 65 phospholipids per protein. No significant change in the fatty acid composition of the disks was observed as a function of change in cholesterol content. This work demonstrates compositional heterogeneity in disk membranes which may ultimately affect function.     

Transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus.

Two phospholipid exchange proteins and two phospholipases C have been employed to determine the phospholipid composition of the outer surface of the membrane of influenza virus. These four protein probes have defined the same accessible and inaccessible pool for each viral phospholipid. Phospholipids which are exchangeable or hydrolyzable are located on the outer surface, whereas the inaccessible pool is located at the inner surface of the viral bilayer. The two pools are unequal in size, with ca. 30% of the total phospholipid accessible to the four proteins, and ca. 70% inaccessible. The membrane is thus highly asymmetric with regard to the amount of phospholipid on each side of the membrane. There is also a marked asymmetry of phospholipid composition. Phosphatidylcholine and phosphatidylinositol are enriched in the outer surface, and sphingomyelim is enriched in the inner surface, whereas phosphatidylethanolamine and phosphatidylserine are present in similar proportions in each surface. This distribution is qualitatively different from that previously reported for the human erythrocyte. The close agreement between results obtained with excahnge proteins and phospholipases C demonstrates that the hydrolytic action of these enzymes does not alter phospholipid asymmetry. The nonperturbing nature of the exchange proteins has permitted the rate of transmembrane movement of phospholipids (flip-flop) in the intact virion to be studied. This process could not be detected after 2 days at 37 degrees C. It was estimated that the half-time for flip-flop is indeterminately in excess of 30 days for sphingomyelin and 10 days for phosphatidylcholine at 37 degrees C. These extremely long times provide a simple explanation for the maintenance of transbilayer asymmetry in influenza virions and possibly, other membranes. Since the viral membrane is acquired by budding through the host cell plasma membrane, the transbilayer distribution of phospholipids observed in the virions presumably reflects a similar asymmetric distribution of phospholipids in the host cell surface membrane. Because animal cells in culture do not incorporate extracellular phospholipid, our results demonstrate that individual cells have the capacity to generate asymmetric membranes.     

Isolation and composition of sheathed flagella from Bdellovibrio bacteriovorus 109J.

A procedure was developed for the purification of sheathed flagella from Bdellovibrio bacteriovorus 109J. Preparations of isolated flagella appeared as filaments 28 nm in diameter, did not vary in sheath content by more than 10% from the mean, and contained 50% protein, 38% phospholipid, and 12% lipopolysaccharide (LPS) by weight. The sheath was readily solubilized by Triton X-100, whether or not EDTA was present, and contained all of the LPS and phospholipid and 30 to 40% of the protein of the intact flagella; sedimentable core filament polypeptides accounted for the remainder. Flagellar LPS was significantly enriched in nonadecenoic acid (19:1) and depleted in beta-hydroxymyristic acid relative to outer membrane LPS from intraperiplasmically grown bdellovibrios. These observations suggest that the sheath is a stable domain distinct from the bulk of the outer membrane. The sheath also contained substantially more phospholipid (57%) and less protein (26%) of a more heterogeneous composition than that of previously described outer membranes. This unusual balance of constituents was predicted to result in a fluid membrane compatible with a model for the generation of motility by rotation of the core filament within a highly flexible sheath.     

Alterations in the Outer Membrane of the Cell Envelope of Heptose-Deficient Mutants of Escherichia coli

The composition of the cell envelope of a heptose-deficient lipopolysaccharide mutant of Escherichia coli, GR467, was studied after fractionation into its outer and cytoplasmic membrane components by means of sucrose density gradient centrifugation. The outer membrane of GR467 had a lower density than that of its parent strain, CR34. Analysis of the fractionated membranes of GR467 indicated that the phospholipid-to-protein ratio had increased 2.4-fold in the outer membrane. The ratio in the mutant cytoplasmic membrane was also increased, although to a lesser extent. By employing a third parameter, the lipid A content of the outer membrane, it was found that the observed phospholipid-to-protein change in the outer membrane was due predominantly to a decrease in the relative amount of protein. This decrease in protein was particularly significant, since it was concomitant with a 68% decrease in the lipid A recovered in the outer membrane of GR467 relative to the lipid A recovered in the outer membrane of CR34. Similar findings were observed in a second heptose-deficient mutant of E. coli, RC-59. The apparent protein deficiency in GR467 was further studied by subjecting solubilized envelope proteins to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that major envelope proteins which were localized in the outer membrane were greatly diminished in GR467. Two revertants of GR467 with the wild-type amounts of heptose had wild-type relative levels of protein in their outer membranes. A partial heptose revertant had a relative level of protein in its outer membrane between those of the mutant and wild type.     

Lipids and NADPH-dependent superoxide production in plasma membrane vesicles from roots of wheat grown under copper deficiency or excess

The effects of in vivo copper on the lipid composition of root plasma membrane and the activities of membrane-bound enzymes, such as NADPH-dependent oxidases and lipoxygenase, were studied. Plants were grown in hydroponic culture for 11 d without Cu supply or in the presence of 50 microM Cu. Control plants were supplied with 0.3 microM Cu. Growth of roots was severely affected in the 50 microM Cu-grown plants, whereas roots grown in Cu-deficient solution did not show any difference in comparison with the control. The 50 microM Cu concentration caused an increase in the leakage of K(+) ions as well. Excess metal supply resulted in a decrease in the total lipid content of plasma membrane, a higher phospholipid amount and a reduction of steryl lipids (free sterols, steryl glycosides and acylated steryl glycosides). Cu depletion in the growth solution had only a slight effect on the plasma membrane lipid composition. In comparison with the control, only the excess of Cu caused a decrease in the lipid to protein ratio as well as a change in the phospholipid composition, with a lower phosphatidylcholine to phosphatidylethanolamine ratio. The degree of unsaturation of root plasma membranes decreased following the 0 Cu treatment and even more after the 50 microM Cu supply. Plasma membranes of wheat grown under metal deficiency and excess showed increased NADPH-dependent superoxide-producing oxidase activities, whereas membrane-bound lipoxygenase was not increased or activated due to Cu treatments. The consequences of changes in plasma membrane lipid composition and activated oxygen production as a result of Cu treatments are discussed.     

MsbA is not required for phospholipid transport in Neisseria meningitidis

The outer membrane of Gram-negative bacteria contains phospholipids and lipopolysaccharide (LPS) in the inner and outer leaflet, respectively. Little is known about the transport of the phospholipids from their site of synthesis to the outer membrane. The inner membrane protein MsbA of Escherichia coli, which is involved in the transport of LPS across the inner membrane, has been reported to be involved in phospholipid transport as well. Here, we have reported the construction and the characterization of a Neisseria meningitidis msbA mutant. The mutant was viable, and it showed a retarded growth phenotype and contained very low amounts of LPS. However, it produced an outer membrane, demonstrating that phospholipid transport was not affected by the mutation. Notably, higher amounts of phospholipids were produced in the msbA mutant than in its isogenic parental strain, provided that capsular biosynthesis was also disrupted. Although these results confirmed that MsbA functions in LPS transport, they also demonstrated that it is not required for phospholipid transport, at least not in N. meningitidis.     

Phosphatidylcholine Asymmetry in Electroplax from the Electric Eel: Use of a Phosphatidylcholine Exchange Protein

Phosphatidylcholine asymmetry in the inner and outer leaflets of the plasma membrane bilayer of the innervated and noninnervated surfaces of the electroplax cell was determined, using a Phosphatidylcholine exchange protein. The exchange protein from bovine liver catalyzed the exchange of Phosphatidylcholine from small unilamellar vesicles to the outer monolayer of the plasma membrane bilayer. The exchange protein did not penetrate to the inner monolayer of the plasma membrane, did not modify the permeability of the electroplax, and did not alter the phospholipid or cholesterol content of the electroplax. In the innervated plasma membrane, 42% of the Phosphatidylcholine is in the outer leaflet, 33% is in the inner leaflet, and 25% is inaccessible to the exchange protein. Corresponding values for the noninnervated plasma membrane are 56, 26, and 18%, respectively. These results are similar to Phosphatidylcholine asymmetry in other biological membranes. This unique cell can be used as a model to test the effects on phospholipid asymmetry of compounds that act on the membrane.     

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.     

Heterogeneity in Lipid Composition of the Outer Membrane and Cytoplasmic Membrane of Pseudomonas BAL-31

The outer membranes and cytoplasmic membranes of the marine bacterium Pseudomonas BAL-31 were separated by washing the cells three times in 0.5 M NaCl and twice in 0.5 M sucrose. Electron microscopy during the removal of membranes revealed that the outer membranes fragmented in a regular manner to give rise to fairly uniform vesicles measuring approximately 140 nm in diameter. Isolated outer membranes had a buoyant density in sucrose of 1.230 g per cm(3), whereas the cytoplasmic membranes had a density of 1.194 g per cm(3). The removal of the outer membrane during the application of this procedure was monitored by measuring the release of 2-keto-3-deoxyoctulosonic acid and phospholipid. The cells lost 85.5% of their 2-keto-3-deoxyoctulosonic acid and 47.3% of their phospholipid during this treatment. Complete recovery of outer membrane material could be achieved. The removal of 25.5% of the 2-keto-3-deoxyoctulosonic acid and 0.9% of the phospholipid rendered the cells sensitive to lysis with Triton X-100. The phospholipid composition of the outer membrane was calculated to be 78.9% phosphatidylethanolamine and 16.1% phosphatidylglycerol. The phospholipid composition of the cytoplasmic membrane proved to be 71.5% phosphatidylethanolamine and 23.5% phosphatidylglycerol. The fatty acid composition was also found to be quantitatively heterogeneous between the two membranes.     

Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay.

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.     

Translocation of phospholipids between the outer and inner membranes of Salmonella typhimurium.

The reversibility and specificity of phospholipid translocation between the inner and outer membrane of Salmonella typhimurium has been investigated by incorporating exogenous lipids from phospholipid vesicles into the outer membrane of intact cells. Translocation of newly incorporated phospholipids to the inner membrane was demonstrated by decarboxylation of vesicle-derived phosphatidylserine and by recovery of vesicle constituents in both inner and outer membrane fractions. All Salmonella phospholipids tested, as well as phosphatidylcholine and cholesteryl oleate were effectively translocated to the inner membrane. However, no translocation of vesicle-derived lipopolysaccharide or an incomplete biosynthetic precursor of lipid A could be detected. Translocation of phospholipids and cholesteryl ester was rapid and extensive, and appeared to lead to equilibration of the lipids between the two membranes. The mechanism of intermembrane translocation has not been established, but the results are suggestive of diffusional flow across zones of adhesion between the inner and outer membranes.     

Regulation of the synthesis of surface protein in the cell cycle of E. coli B/r.

We have studied the biogenesis of the envelope of E. coli B/r by measuring the synthesis of protein in separated inner and outer membranes during the cell cycle. While total protein and bulk inner membrane protein were synthesized continuously and at an exponentially increasing rate throughout the cycle, bulk outer membrane protein was synthesized at a constant rate throughout the cycle with an abrupt doubling in rate occurring 10–15 min before division. A similar pattern was observed when the rate of synthesis of an individual protein, the 36.5K outer membrane protein, was measured directly in total cell lysates. Neither thymine starvation nor changes in gene dosage of exponential cultures affected the synthesis of outer membrane protein, indicating that the doubling in rate is not controlled by a gene duplication mechanism. Other findings, however, further indicate that outer membrane protein synthesis is regulated in some way. Thus the concentration of 36.5K porin per unit surface area remained constant as the surface area/volume ratio varied widely with growth rate. We also obtained direct evidence for an overall limitation on the rate of synthesis of bulk outer membrane proteins; when a new class of outer membrane proteins was induced, the rate of synthesis of other surface proteins was correspondingly reduced. On the basis of these results, we discuss a model in which the linear growth of outer membrane protein results from a limitation of outer membrane polypeptide synthesis at the translational level, reflecting the linear expansion of the underlying peptidoglycan layer in the envelope.