Membranes Of Wild-type Cells Research Articles

Slc44a2 deletion alters tetraspanin and N-cadherin expression: Reduced adhesion and enhanced proliferation in cultured mesenchymal lung cells

Slc44a2 is reported to interact with tetraspanins CD9 and CD81. To investigate how Slc44a2 affects adhesion protein expression, cells from wild-type (WT) Slc44a2+/+, heterozygous (HET) Slc44a2+/-, and knockout (KO) Slc44a2-/- mice were cultured from lung tissue. The cultured cells expressed vimentin, N-cadherin, p120 catenin, beta-catenin, actin, CD9, and CD81, but not E-cadherin. Vimentin expression with lack of E-cadherin indicated that the cultured cells were of mesenchymal origin. Slc44a2 KO cells and HET cells demonstrated lower adherence and faster proliferation than the WT cells. All three groups displayed dramatically altered intracellular distribution of N-cadherin, CD9, and CD81. The CD9 membrane foci observed in WT cell membranes were less frequent and diminished in size in HET cells and KO cells. N-cadherin was dispersed throughout both the cytoplasm and membrane in WT cells, with similar yet weaker distribution in HET cells; however, in KO cells, N-cadherin was densely aggregated in the perinuclear cytoplasm. CD81 had a distribution pattern in WT, HET, and KO cells similar to that of N-cadherin with dense cytoplasmic clusters in the cells. KO cells also exhibited reduced filamentous actin as compared to WT cells. These results suggest that Slc44a2 is necessary for proper cellular localization of adhesion proteins and growth regulation that may be related to altered adhesion signals.

Protein Implicated in Nonsyndromic Mental Retardation Regulates Protein Kinase A (PKA) Activity

Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacity to form dendrites and synapses in culture. At the biochemical level, CC2D1A transduces signals to the cyclic adenosine 3',5'-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation.

Overexpression of VMAT-2 and DT-diaphorase protects substantia nigra-derived cells against aminochrome neurotoxicity

We tested the hypothesis that both VMAT-2 and DT-diaphorase are an important cellular defense against aminochrome-dependent neurotoxicity during dopamine oxidation. A cell line with VMAT-2 and DT-diaphorase over-expressed was created. The transfection of RCSN-3 cells with a bicistronic plasmid coding for VMAT-2 fused with GFP-IRES-DT-diaphorase cDNA induced a significant increase in protein expression of VMAT-2 (7-fold; P<0.001) and DT-diaphorase (9-fold; P<0.001), accompanied by a 4- and 5.5-fold significant increase in transport and enzyme activity, respectively. Studies with synaptic vesicles from rat substantia nigra revealed that VMAT-2 uptake of 3H-aminochrome 6.3±0.4nmol/min/mg was similar to dopamine uptake 6.2±0.3nmol/min/mg that which were dependent on ATP. Interestingly, aminochrome uptake was inhibited by 2μM lobeline but not reserpine (1 and 10μM). Incubation of cells overexpressing VMAT-2 and DT-diaphorase with 20μM aminochrome resulted in (i) a significant decrease in cell death (6-fold, P<0.001); (ii) normal ultra structure determined by transmission electron microscopy contrasting with a significant increase of autophagosome and a dramatic remodeling of the mitochondrial inner membrane in wild type cells; (iii) normal level of ATP (256±11μM) contrasting with a significant decrease in wild type cells (121±11μM, P<0.001); and (iv) a significant decrease in DNA laddering (21±8pixels, P<0.001) cells in comparison with wild type cells treated with 20μM aminochrome (269±9). These results support our hypothesis that VMAT-2 and DT-diaphorase are an important defense system against aminochrome formed during dopamine oxidation.

Furrow-like invaginations of the yeast plasma membrane correspond to membrane compartment of Can1

Plasma membrane of the yeast Saccharomyces cerevisiae contains stable lateral domains. We have investigated the ultrastructure of one type of domain, the membrane compartment of Can1 (MCC). In two yeast strains (nce102Delta and pil1Delta) that are defective in segregation of MCC-specific proteins, we found the plasma membrane to be devoid of the characteristic furrow-like invaginations. These are highly conserved plasma membrane structures reported in early freeze-fracture studies. Comparison of the results obtained by three different approaches - electron microscopy of freeze-etched cells, confocal microscopy of intact cells and computer simulation - shows that the number of invaginations corresponds to the number of MCC patches in the membrane of wild-type cells. In addition, neither MCC patches nor the furrow-like invaginations colocalized with the cortical ER. In mutants exhibiting elongated MCC patches, there are elongated invaginations of the appropriate size and frequency. Using various approaches of immunoelectron microscopy, the MCC protein Sur7, as well as the eisosome marker Pil1, have been detected at these invaginations. Thus, we identify the MCC patch, which is a lateral membrane domain of specific composition and function, with a specific structure in the yeast plasma membrane - the furrow-like invagination.

Erv14 family cargo receptors are necessary for ER exit during sporulation inSaccharomyces cerevisiae

Sporulation of Saccharomyces cerevisiae is a developmental process in which four haploid spores are created within a single mother cell. During this process, the prospore membrane is generated de novo on the spindle pole body, elongates along the nuclear envelope and engulfs the nucleus. By screening previously identified sporulation-defective mutants, we identified additional genes required for prospore membrane formation. Deletion of either ERV14, which encodes a COPII cargo receptor, or the meiotically induced SMA2 gene resulted in misshapen prospore membranes. Sma2p is a predicted integral membrane that localized to the prospore membrane in wild-type cells but was retained in the ER in erv14 cells, suggesting that the prospore membrane morphology defect of erv14 cells is due to mislocalization of Sma2p. Overexpression of the ERV14 paralog ERV15 largely suppressed the sporulation defect in erv14 cells. Although deletion of ERV15 alone had no phenotype, erv14 erv15 double mutants displayed a complete block of prospore membrane formation. Plasma membrane proteins, including the t-SNARE Sso1p, accumulated in the ER upon transfer of the double mutant cells to sporulation medium. These results reveal a developmentally regulated change in the requirements for ER export in S. cerevisiae.

A Role for Pet100p in the Assembly of Yeast Cytochrome c Oxidase: INTERACTION WITH A SUBASSEMBLY THAT ACCUMULATES IN A pet100 MUTANT

The biogenesis of multimeric protein complexes of the inner mitochondrial membrane in yeast requires a number of nuclear-coded ancillary proteins. One of these, Pet100p, is required for cytochrome c oxidase. Previous studies have shown that Pet100p is not required for the synthesis, processing, or targeting of cytochrome c oxidase subunits to the mitochondrion nor for heme A biosynthesis. Here, we report that Pet100p does not affect the localization of cytochrome c oxidase subunit polypeptides to the inner mitochondrial membrane but instead functions after they have arrived at the inner membrane. We have also localized Pet100p to the inner mitochondrial membrane in wild type cells, where it is present in a subassembly (Complex A) with cytochrome c oxidase subunits VII, VIIa, and VIII. Pet100p does not interact with the same subunits after they have been assembled into the holoenzyme. In addition, we have identified two subassemblies that are present in pet100 null mutant cells: one subassembly (Complex A') is composed of subunits VII, VIIa, and VIII but not Pet100p, and another subassembly (Complex B) is composed of subunits Va and VI. Because pet100 null mutant cells lack assembled cytochrome c oxidase but accumulate Complexes A' and B it appears likely that these subassemblies of cytochrome c oxidase subunits are intermediates along an assembly pathway for holocytochrome c oxidase and that Pet100p functions in this pathway to facilitate the interaction(s) between Complex A' and other cytochrome c oxidase subassemblies and subunits.

Rpn5 Is a Conserved Proteasome Subunit and Required for Proper Proteasome Localization and Assembly

Proper function of the 26 S proteasome requires assembly of the regulatory complex, which is composed of the lid and base subcomplexes. We characterized Rpn5, a lid subunit, in fission yeast. We show that Rpn5 associates with the proteasome rpn5. Deletion (rpn5Delta) exacerbates the growth defects in proteasome mutants, leading to mitotic abnormalities, which correlate with accumulation of polyubiquitinated proteins, such as Cut2/securin. Rpn5 expression is tightly controlled; both overexpression and deletion of rpn5 impair proteasome functions. The proteasome is assembled around the inner nuclear membrane in wild-type cells; however, in rpn5Delta cells, proteasome subunits are improperly assembled and/or localized. In the lid mutants, Rpn5 is mislocalized in the cytosol, while in the base mutants, Rpn5 can enter the nucleus, but is left in the nucleoplasm, and not assembled into the nuclear membrane. These results suggest that Rpn5 is a dosage-dependent proteasome regulator and plays a role in mediating proper proteasome assembly. Moreover, the Rpn5 assembly may be a cooperative process that involves at least two steps: 1) nuclear import and 2) subsequent assembly into the nuclear membrane. The former step requires other components of the lid, while the latter requires the base. Human Rpn5 rescues the phenotypes associated with rpn5Delta and is incorporated into the yeast proteasome, suggesting that Rpn5 functions are highly conserved.

Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells.

The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a approximately 95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).

Purified components of the Escherichia coli Tat protein transport system form a double-layered ring structure.

The Escherichia coli twin arginine translocation (Tat) system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. The genes tatA, tatB, tatC and tatE code for integral membrane proteins that are components of the Tat pathway. Cells co-overexpressing tatABCDE show an increased rate of export of a signal peptide-defective Tat precursor protein and a complex containing the TatA and TatB proteins can be purified from the membranes of such cells. The purified TatAB complex has an apparent molecular mass of 600 kDa as measured by gel permeation chromatography and, like the membranes of wild-type cells, contains a large molar excess of TatA over TatB. Negative stain electron microscopy of the complex reveals cylindrical structures that may correspond to the Tat protein transport channel.

Characterization of fatty acid composition, spore germination, and thermal resistance in a nisin-resistant mutant of Clostridium botulinum 169B and in the wild-type strain.

The membrane fatty acids, thermal resistance, and germination of a nisin-resistant (Nisr) mutant of Clostridium botulinum 169B were compared with those of the wild-type (WT) strain. In the membranes of WT cells, almost 50% of the total fatty acids were unsaturated, but in those of Nisr cells, only 23% of the fatty acids were unsaturated. WT and Nisr spores contained similar amounts (approximately 23%) of unsaturated fatty acids, but the saturated straight-chain/branched-chain ratio was significantly higher in Nisr spores than in WT spores. These fatty acid differences suggest that Nisr cell and spore membranes may be more rigid, a characteristic which would interfere with the pore-forming ability of nisin. Nisr C. botulinum did not produce an extracellular nisin-degrading enzyme, nor were there any differences in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of coat proteins extracted from WT and Nisr spores, eliminating these as possible reasons for nisin resistance. Nisr spores had thermal resistance parameters similar to those of WT spores. In WT spores, but not in Nisr spores, nisin caused a 40% reduction in thermal resistance and a twofold increase in the germination rate. Because the nisin-induced increase in the germination rate of WT spores occurred only in the presence of a germinant (a molecule that triggers germination), nisin can be classified as a progerminant (a molecule that stimulates germination only in the presence of a germinant).

Light-induced and osmotically-induced changes in chlorophyll a fluorescence in two Synechocystis sp. PCC 6803 strains that differ in membrane lipid unsaturation

Membranes of wild-type (WT) cells of the cyanobacterium Synechocystis sp. PCC 6803 are abundant in polyunsaturated fatty acids in membrane lipids and thus more fluid than membranes of desA-/desD- mutant cells which contain no polyunsaturated fatty acids. Using intact cells we examined the effects of normal and chilling temperatures on membrane fluidity-dependent properties. We probed the thylakoid membranes by inducing light/dark acclimative changes in chlorophyll a (Chl a) fluorescence; and we probed the plasma membranes either by suppressing the Chl a fluorescence of light-acclimated cells under hyper-osmotic conditions, or by measuring the electric conductivity of cell suspensions. Thylakoid membranes of mutant cells undergo reversible thermotropic transition between 19 °C and 22 °C (midpoint at 20.5 °C). No analogous transition was detected in the thylakoid membranes of WT cells in the temperature range from 2 to 34 °C. Plasma me mbranes of both WT and mutant cells did not experience thermotropic transition in the temperature range from 2 °C to 34 °C as detected either fluorimetrically or by means of electric conductivity. Hyper-osmotic conditions caused fast transient fluorescence quenching in WT cells at 34 °C, but not at 14 °C, and not in mutant cells at either 34 °C or 14 °C. This transient quenching sensed probably the higher fluidity of the plasma membranes of WT cells. Hyper-osmotic media and dark acclimation had similar effects on the 77 K fluorescence of Synechocystis cells: they suppressed the ratio of photosystem II fluorescence to photosystem I fluorescence.

Submitochondrial distributions and stabilities of subunits 4, 5, and 6 of yeast cytochrome oxidase in assembly defective mutants

The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex. All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria. Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover. As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction. A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a. In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a. The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6. The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3. Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.

The architecture of unusual membrane tubes in the B800–850 light-harvesting bacteriochlorophyll-deficient mutant 19 ofRhodobacter sphaeroides

Abstract The B800–850 light-harvesting bacteriochlorophyll-deficient mutant 19 of Rhodobacter sphaeroides exhibits in ultrathin sections not only regularly shaped intracytoplasmic membrane (ICM) vesicles (diameter of 43 nm), but also long tubes of ICM with diameters ranging from 60 to 95 nm. Freeze-fracture preparations revealed that these tubes were densely covered with intramembrane particles of 10.4 nm in diameter. According to the ratio of protein to phospholipid as calculated for the ICM tubes of mutant 19 these tubes do not represent lipid-enriched ICM-invaginations as discussed for other mutants of R. sphaeroides with similar tubular ICM. A comparable particle arrangement was found on the plasmic fracture face of the cytoplasmic membrane in wild-type cells of R. sphaeroides . The architecture of the ICM in the B800–850-deficient mutant Ala + of the closely related R. capsulatus exhibited, in comparison to the wild-type strain, significant differences in the number of particles per μum 2 and in the ratio of protein to phospholipid but no significant differences in the size of the intramembrane particles.

Multidrug resistance in MCF-7 human breast cancer cells is associated with increased expression of nucleoside transporters and altered uptake of adenosine

The rate of adenosine uptake and the corresponding expression of nucleoside transporters were studied in several MCF-7 human breast-cancer cell lines that express different levels of multidrug resistance (MDR). Kinetic studies of adenosine transport in these cell lines revealed that the mean apparent Km and Vmax values for the nucleoside transporters increased with increasing MDR. The apparent Km and the apparent Vmax of Adriamycin-resistant (ADR10) cell lines were respectively 3.2- and 1.8- fold those of Adriamycin-sensitive wild-type (WT) cells (P less than 0.001). A partially revertant cell line (ADR10rev) that was derived from the ADR10 line and was partially sensitive to Adriamycin exhibited apparent Km and Vmax parameters that lay between those of the ADR10 and WT cells (P less than 0.001 vs ADR10 cells; P less than 0.05 vs WT cells). ADR10 cell membranes bound greater than 4 times more of the nucleoside transporter blockers [3H]-nitrobenzylthioinosine [( 3H]-NBI) and [3H]-dipyridamole [( 3H]-DPR) than did WT cell membranes per unit protein (P less than 0.0001). Scatchard analysis revealed a 2-3 times greater density for nucleoside transporters in ADR10 membranes as compared with those in WT membranes. ADR10rev membranes bound less [3H]-NBI and [3H]-DPR than did ADR10 membranes (P less than 0.001), but they bound more of the blockers than did WT membranes (P less than 0.05). A 2.5-h exposure to 200 nM phorbol-12,13-dibutyrate (PDBu), which activates protein kinase C (PKC) and induces WT cells to exhibit a 4-fold increased transient MDR phenotype, increased the apparent Km of WT cells for adenosine transport by greater than 2 times (P less than 0.001) to a value close to that found for the ADR10 cells. An identical exposure of ADR10 cells to PDBu produced no significant effect. The apparent Km of ADR10rev cells was increased 1.4 times by a 2.5-h PDBu exposure. None of the cell lines were affected by a 2.5-h exposure to 200 nM phorbol-13,10-diacetate (PDA), a much less active phorbol, or vehicle. These results suggest that MDR in MCF-7 cells is associated with changes in nucleoside transport, including both the number of transporters and their rate of transport, and that such changes can be partially mimicked by stimulation of PKC.

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.

Accumulation and secretion of exoglucanase activity in yeast secretory mutants

Representative conditional yeast secretory mutants, blocked in transport of secretory and plasma membrane proteins from the endoplasmic reticulum (sec 18), from the Golgi body (sec 7) and in transport of secretory vesicles (sec 1), accumulated exoglucanase, a constitutive yeast activity, when incubated at the restrictive temperature (37 degrees C). Different proportions of the accumulated activity were released by mutant cells under permissive conditions. The presence or absence of cycloheximide during the secretion period made no differences in the results. More than 90% of the internal activity was bound to membrane in wild type cells. However, only the soluble pool underwent changes during the accumulation or secretion periods. The bulk of secretory invertase accumulated by sec 1 was also soluble. By contrast sec 7 and sec 18 accumulated membrane-bound as well as soluble invertase forms and both were secreted in similar proportions in each mutant. More than 90% of the accumulated invertase was secreted at the permissive temperature in sec 18 cells. That percentage was significantly lower for exoglucanase (less than 65%). Concomitantly, invertase accumulated by this mutant exited from the cells with a lower half time (t1/2 = 24 min) than accumulated exoglucanase (t1/2 = 150 min). These results may be interpreted assuming that exoglucanase is exported by a passive flow of the soluble pool.

New mechanism for post-translational processing during assembly of a cytoplasmic membrane protein?

Insertion of nitrate reductase into the Escherichia coli cytoplasmic membrane was examined by following the fate of pulse-labeled enzyme in both the membrane and cytoplasm during various times after the addition of an unlabeled chase. The polypeptide composition of this labeled enzyme was determined by autoradiography of immunoprecipitated material after separation on sodium dodecyl sulfate-polyacrylamide gels. The data presented here indicate that immediately after appropriate insertion of the enzyme into the membrane, a post-translational event occurs which converts the cytoplasmically synthesized form of subunit B (B') to the form found in the completely assembled enzyme (B). B' is distinguished from B by its more rapid electrophoretic mobility. B' was found in the cytoplasm of all strains tested, in the membrane of strains with defects in enzyme insertion (hemA and chlE), and as a transient component in the membrane of wild-type cells.

Reduced synthesis of [14C]mannosyl oligosaccharide-lipid by membranes prepared from concanavalin A-resistant Chinese hamster ovary cells.

We compared the synthesis of mannosyl oligosaccharide-lipid by membranes of wild type and concanavalin A-resistant Chinese hamster ovary cells during incubations with GDP-[14C]mannose and UDP-N-acetylglucosamine. The membranes prepared from exponentially growing mutant cells incorporated 10- to 70-fold less [14C]mannose into oligosaccharide-lipid than did membranes of wild type cells. The lectin-resistant cells are temperature-sensitive for growth. Using temperature-resistant revertants, we showed that the reduction in labeled oligosaccharide-lipid correlated with resistance to concanavalin A rather than with the temperature-sensitive phenotype. Tunicamycin, a specific inhibitor of oligosaccharide-lipid synthesis, reduced the synthesis of the oligosaccharide-lipid by membranes of wild type cells but did not affect the residual synthesis measured in membranes of the mutant cells. As the mutant cells were grown to high density, there was a marked increase in the synthesis of oligosaccharide-lipid by the membranes.