Characteristics and biosynthesis of membrane proteins of lipid bodies in the scutella of maize (<i>Zea mays L</i>.)

Qu, Rongda; Wang, Shue-Mei; Lin, Yon-Hui; Vance, Vicki; Huang, Anthony H. C.

doi:10.1042/bj2350057

Cited by 91 publications

(44 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Enlargement ofthe vesicle eventually produces a lipid body which is composed of a bag of triacylglycerol surrounded by a half-unit membrane of phospholipids and proteins. Earlier, we showed that the lipid body membrane proteins are synthesized on the RER (16). The localization of diacylglycerol acyltransferase, the last enzyme of triacylglycerol biosynthesis, in the ER further supports the ER vesiculation model.…”

supporting

confidence: 76%

Diacylglycerol Acyltransferase in Maturing Oil Seeds of Maize and Other Species

Cao

Huang²

1986

Plant Physiol.

View full text Add to dashboard Cite

Diacylglycerol acyltransferase (EC 2.3.1.20) activity was detected in the microsomal fractions of maturing maize scutellum, soybean cotyledon, peanut cotyledon, and castor bean endosperm. The activity detected was high enough to account for the in vivo rate of triacylglycerol synthesis. The activity of the maize enzyme was characterized using diolein micelles prepared by sonication in Tween 20 as the substrate. The activity was highest at pH values of 6 to 7. The activity was proportional to the amount of enzyme added, and the reaction rate was linear for about 2 minutes. The enzyme was not inactivated by Tween 20, Zwitterion 3-08, Triton-X 100, and cholate, but was inactivated completely by sodium dodecyl sulfate. The enzyme was active on linoleoyl coenzyme A (CoA), palmitoyl CoA, and oleoyl CoA, although the activity was highest on linoleoyl CoA. Endogenous diacylglycerol was present in the microsomes, and the enzyme activity was only partially dependent on the addition of external diolein. Subcellular fractionation of the total scutellum extract in sucrose density gradients was performed. By comparing the migration of the enzyme between rate and equilibrium centrifugation, and between equilibrium centrifugation in the presence and absence of magnesium ions in the preparative media, the enzyme was shown to be associated with the rough endoplasmic reticulum. Some of the above findings on the maize enzyme were extended to the enzymes from castor bean, soybean, and peanuts.Diacylglycerol acyltransferase (EC 2.3.1.20) catalyzes the final step in the synthesis of triacylglycerols in oil seeds (18,22). It is also the only known enzyme unique to the long biosynthetic pathway of triacylglycerols, since the diacylglycerol produced could also be used to produce phospholipids or galactolipids. In spite of the importance of this enzyme in triacylglycerol biosynthesis in oil seeds, its properties have not been well studied.Diacylglycerol acyltransferase in the microsomal fraction of developing seeds has been assayed directly or together with other enzymes (6,10,20,21). In a recent, and so far the most detailed, study, the general properties of the enzyme in a safflower microsomal fraction were characterized (10). However, the detected level ofactivity was almost a magnitude lower than that required to catalyze the sequence of reaction from glycerol phosphate to triacylglycerol (21, 22). Using an enzyme assay which had been used successfully to study the enzyme in spinach leaves, Martin and Wilson (13) failed to detect enzyme activity in the cotyledon extract of developing soybean.An important but unknown aspect of diacylglycerol acyltransferase is its subcellular location. In general, the microsomal fractions were used to study the enzyme activity, and they 'Supported by National Science Foundation grant DMB 85-15556.presumably contained vesicles of the ER as well as membranes of other subcellular particles, including broken plastids. In a recent detailed analysis of the subcellular location ofthe enzyme in spinach...

show abstract

supporting

confidence: 76%

Diacylglycerol Acyltransferase in Maturing Oil Seeds of Maize and Other Species

Cao

Huang²

1986

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…These lipid bodies contain a limited number of distinct proteins which are believed to be integrated in the surrounding monolayer (11). The protein termed L3 (Mr 16,500) and lipase (Mr 65,000) are the best studied (8,9,11,13 Plant Materials. Kernels of inbred maize (Zea mays L. cv Mo 17) were obtained from the Illinois Foundation Seed Corp., (Champaign, IL).…”

Section: Methodsmentioning

confidence: 99%

“…The lipid bodies in scutella of maize kernels have been studied the most extensively. These lipid bodies contain a limited number of distinct proteins which are believed to be integrated in the surrounding monolayer (11). The protein termed L3 (Mr 16,500) and lipase (Mr 65,000) are the best studied (8,9,11,13 Plant Materials.…”

Section: Methodsmentioning

confidence: 99%

“…To check the specificity of labeling, the following series of controls were performed: (a) L3 antibodies were replaced with normal (nonimmune) rabbit serum, (b) L3 antibodies were replaced with antibodies directed against the unrelated Chll-protein CPII* of thylakoid membranes (3), (c) L3 antibodies were preabsorbed with lipid body membrane proteins isolated from ungerminated maize kernels (0.5 mg/ml) (11) Homogenates of scutella from ungerminated maize kernels can be resolved into a large number of protein bands using SDS-PAGE (Fig. 1).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Immunogold Localization of the L3 Protein of Maize Lipid Bodies during Germination and Seedling Growth

Fernandez¹,

Qu²,

Huang³

et al. 1988

Plant Physiol.

View full text Add to dashboard Cite

ABSTRACrWe have used antibodies directed against the 16.5 kilodalton protein L3, the most abundant integral protein of maize (Zea mays L. cv Mo 17) lipid bodies, to follow the fate of this protein in scutellar parenchyma cells of maize during germination and subsequent seedlig growth. Using gel electrophoresis and immunoblotting as well as immunocytochemical electron microscopy, we found that the amount of L3 decreases gradually during the first 3 to 4 days of seling growth and more rapidly over the course of the next several days. Immunogold localization of the protein on thin sections indicated that L3 is found exclusively in the surface phospholipid monolayer of lipid bodies. The density of L3 in the surface layer of individual lipid bodies does not change during seedling growth; therefore, the decrease in the amount of L3 can be attributed to a decrease in the number of lipid bodies rather than to selective removal of protein components from the surface of all lipid bodies. Thus, L is apparently degraded at the same time as the matrix lipid of each lipid body. Unlike lipase, L3 does not appear to be transferred to other cellular compartments such as vacuoles during late stages of seedling growth.The level of activity gradually increases, peaks at d 5 and then remains high for at least 5 more d (13). The distribution of the enzyme in cells has been studied using cellular fractionation techniques. Lipase activity is found initially (3-4 d) in the lipid body fraction but appears to shift into other cellular compartments, possibly vacuoles, at later stages (6-10 d) (13). In contrast to the lipase data, no information on the distribution of L3 in maize scutellar cells during germination and subsequent seedling growth is available. In this paper, we report on the immunological localization of the L3 protein and an investigation of its fate following germination. Our data indicate that the amount of L3 decreases gradually over the first 4 d and then decreases dramatically over the course of the next 3 d. Thus, unlike lipase, L3 is apparently degraded at the same time as the matrix lipid of the lipid bodies. In addition, L3 is confined exclusively to the surface of lipid bodies, and, unlike lipase, does not appear to be transfered to other cytoplasmic compartments during later stages of seedling growth. MATERIALS AND METHODSThe triacylglycerols stored in the lipid bodies of seeds are degraded during germination to provide energy and precursor molecules for embryo growth (2, 6). Despite the importance of this process, little is known on a cellular level about the fate of the lipid bodies, and, after the matrix lipid has been depleted, the fate of the components of the phospholipid monolayers that surround the hydrophobic core (10, 16). The lipid bodies in scutella of maize kernels have been studied the most extensively. These lipid bodies contain a limited number of distinct proteins which are believed to be integrated in the surrounding monolayer (11). The protein termed L3 (Mr 16,500) and lipase (Mr 65,000) are t...

show abstract

“…Current evidence suggests that oleosin targets to the ER by an SRP-mediated pathway; oleosin mRNA is found in ERbound polyribosomes (27), and exogenous SRP inhibits oleosin translation in vitro (28). Oleosin expression in yeast mutants also indicates a requirement for SRP and Sec61 (29).…”

mentioning

confidence: 99%

Membrane Protein Topology of Oleosin Is Constrained by Its Long Hydrophobic Domain

Abell

High

Moloney

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Oleosin proteins from Arabidopsis assume a unique endoplasmic reticulum (ER) topology with a membraneintegrated hydrophobic (H) domain of 72 residues, flanked by two cytosolic hydrophilic domains. We have investigated the targeting and topological determinants present within the oleosin polypeptide sequence using ER-derived canine pancreatic microsomes. Our data indicate that oleosins are integrated into membranes by a cotranslational, translocon-mediated pathway. This is supported by the identification of two independent functional signal sequences in the H domain, and by demonstrating the involvement of the SRP receptor in membrane targeting. Oleosin topology was manipulated by the addition of an N-terminal cleavable signal sequence, resulting in translocation of the N terminus to the microsomal lumen. Surprisingly, the C terminus failed to translocate. Inhibition of C-terminal translocation was not dependent on either the sequence of hydrophobic segments in the H domain, the central proline knot motif or charges flanking the H domain. Therefore, the topological constraint results from the length and/or the hydrophobicity of the H domain, implying a general case that long hydrophobic spans are unable to translocate their C terminus to the ER lumen.Membrane proteins targeted to the ER 1 generally display a uniform and consistent topology, in which the orientation of membrane spans is determined by the interaction between nascent polypeptides and the translocon (1, 2). A set of general rules has been established that are useful for prediction of unknown topologies. These rules also further our understanding of the underlying mechanisms of membrane insertion. In the simplest case, individual membrane spans can be orientated by sequential insertion, resulting in adoption of the opposite orientation to the previous span (3). The presence of charged residues in flanking regions is a critical influence on the orientation of individual membrane spans; positively charged residues show a strong preference for retention in the cytosol, while negatively charged residues have a weak affinity for translocation to the ER lumen (4 -7). Variants of a yeast protein, Ste2p, a G protein-coupled pheromone receptor, strictly followed the "positive inside" rule for the first transmembrane span (8). The N terminus is translocated only if the charge difference is reversed by removal of all the N-terminal positive charges or addition of C-terminal negative charges. ER membrane does not support a transmembrane potential, suggesting that this preference may be due to charged residues interacting with phospholipids (9). In the case of signal-anchors, translocation of the N terminus can be blocked by the adoption of a fully folded conformation (10). Prediction of topology can also be aided by analyzing the membrane span itself. Transmembrane spans up to 25 leucine residues increasingly favored a topology with the N terminus in the lumen as the polyleucine chain was lengthened (11). The interaction between multiple topology determinants is ...

show abstract

Characteristics and biosynthesis of membrane proteins of lipid bodies in the scutella of maize (Zea mays L.)

Cited by 91 publications

References 23 publications

Diacylglycerol Acyltransferase in Maturing Oil Seeds of Maize and Other Species

Diacylglycerol Acyltransferase in Maturing Oil Seeds of Maize and Other Species

Immunogold Localization of the L3 Protein of Maize Lipid Bodies during Germination and Seedling Growth

Membrane Protein Topology of Oleosin Is Constrained by Its Long Hydrophobic Domain

Contact Info

Product

Resources

About