The protein-body proteins phytohemagglutinin and tonoplast intrinsic protein are targeted to vacuoles in leaves of transgenic tobacco

To demonstrate the relationship between protein-bodies in seeds and vacuoles in other tissues, we expressed the coding sequences of two bean (Phaseolus vulgaris L.) protein-body proteins in tobacco (Nicotiana tabacum L.). We chose phytohemagglutinin-L (PHA-L) and tonoplast intrinsic protein (TIP) as representatives of the protein-body contents and protein-body membrane, respectively. The location of the two proteins in the leaves of transgenic tobacco was examined by immunocytochemistry and in preparations of isolated vacuoles. Tonoplast intrinsic protein accumulates primarily in tonoplasts in tobacco leaves, whereas PHA is found exclusively in the vacuolar sap, showing that the signals that target proteins to protein-bodies and their limiting membranes in seeds are correctly recognized in leaves. This observation provides further evidence that proteinbodies of dicotyledonous seeds should be considered as protein-storage vacuoles.Abbreviations TIP tonoplast intrinsic protein - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work has been supported by a grant from the U.S. Department of Agriculture Competitive Research Grants Office to Maarten J. Chrispeels. Herman Höfte is a recipient of a Postdoctoral Fellow-ship from the European Molecular Biology Organization. Craig Dickinson is a recipient of a National Institutes of Health Postdoctoral Fellowship. Loîc Faye was on leave from the Centre National de la Recherche Scientifique, UA203, Université de Rouen, Mont Saint Aignan France and supported by a cooperative CNRS — National S cience Foundation grant. The mention of vendor or product in this paper does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned. We thank Jürgen Denecke for providing plasmid pDE1001, Antje von Schaewen for the plant expression cassette and Marie-Theres Hauser for the modified vacuole preparation protocol.     

Synthesis of an integral protein of the protein-body membrane in Phaseolus vulgaris cotyledons

Protein-body membranes (PBMs) were isolated from cotyledons of Phaseolus vulgaris L. by a procedure involving osmotic shock of purified protein bodies. The purified PBMs have a characteristic density of 1.16 g cm^-3. Treatment of the membranes with increasing concentrations of detergent (Triton X-100) or with a solution at pH 12.0 showed that the membranes contained a characteristic integral protein (IMP) with a relative molecular mass of 25,000. This IMP is not a glycoprotein. When developing cotyledons were labeled with ³H-amino acids for 2–3 h, a radioactive polypeptide with the same mobility on denaturing polyacrylamide gels as IMP was found to be associated with the rough endoplasmic reticulum (ER). During a 24-h chase, a considerable portion of the radioactivity slowly transferred into the IMP associated with more rapidly sedimenting organelles, which sedimented in the same region of the sucrose gradients as the PBMs. Antibodies prepared against purified IMP crossreacted with an ER-associated protein which had the same mobility on denaturing acrylamide gels as authentic IMP. Synthesis of IMP occurred at all stages of cotyledon development examined, but not during seed germination. The results show that a newly synthesized protein of the PBM is associated with the rough ER, just like the soluble matrix proteins, phaseolin (R. Bollini, W. Van der Wilden and M.J. Chrispeels, 1983, J. Cell Biol. 96,999–1007) and phytohemagglutinin (M.J. Chrispeels and R. Bollini, 1982, Plant Physiol. 70, 1425–1428), but that the chase-out from the ER is much slower for IMP than for the matrix proteins.Abbreviations EDTA ethylenediamino-tetraacetic acid - ER endoplasmic reticulum - IMP integral membrane protein - PB protein body - PBM protein-body membrane - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Biogenesis of protein bodies by budding from vacuoles in developing pumpkin cotyledons

Summary Vacuoles were isolated from pumpkin cotyledons at three developmental stages and judged to be pure by light microscopic inspection and marker enzyme assays. The time sequence of structural changes of vacuoles were examined by light microscopic inspection in parallel with their stainability with neutral red. Vacuoles isolated from the early stage of cotyledon development were heterogeneous in size (Ø=2–10 m) but stained uniformly with the dye. In contrast, vacuoles isolated from the middle stage were much larger (Ø=5–15 m), and there exist one to three cores, unstainable with neutral red, within a single vacuole. Electron microscopic observation confirms that vacuoles contain a few protein cores in cotyledon cells at the middle stage. Characteristically at this stage, it was observable that some large cores (Ø=4m) were budding from vacuoles. At the late stage, size of vacuoles becomes much smaller (Ø=6m), nearly equal to that of the protein bodies in dry seeds. Importantly, at this stage most of the volume of each vacuole was occupied by a single core, and only a small matrix space was stainable with neutral red. Suborganellar fractionation indicates that the vacuolar cores were identical to the crystalloids deposited in the protein bodies in dry seeds. Overall results strongly provide the evidence that one crystalloid buds from the vacuole during the later stage of seed maturation, giving rise to a protein body.     

Characterization of two integral membrane proteins located in the protein bodies of pumpkin seeds

Two integral membrane proteins, MP28 and MP23, were found in protein bodies isolated from pumpkin (Cucurbita sp.) seeds. Molecular characterization revealed that both MP28 and MP23 belong to the seed TIP (tonoplast intrinsic protein) subfamily. The predicted 29 kDa precursor to MP23 includes six putative membrane-spanning domains, and the loop between the first and second transmembrane domains is larger than that of MP28. The N-terminal sequence of the mature MP23 starts from residue 66 in the first loop, indicating that an N-terminal 7 kDa fragment that contains one transmembrane domain is post-translationally removed. During maturation of pumpkin seeds, mRNAs for MP28 and MP23 became detectable in cotyledons at the early stage, and their levels increased slightly until a rapid decrease occurred at the late stage. This is consistent with the accumulation of the 29 kDa precursor and MP28 in the cotyledons at the early stage. By contrast, MP23 appeared at the late stage simultaneously with the disappearance of the 29 kDa precursor. Thus, it seems possible that the conversion of the 29 kDa precursor to the mature MP23 might occur in the vacuoles after the middle stage of seed maturation. Both proteins were localized immunocytochemically on the membranes of the vacuoles at the middle stage and the protein bodies at the late stage. These results suggest that both MP28 and the precursor to MP23 accumulate on vacuolar membranes before the deposition of storage proteins, and then the precursor is converted to the mature MP23 at the late stage. These two TIPs might have a specific function during the maturation of pumpkin seeds.     

Molecular characterization of a cold-induced plasma membrane protein gene from wheat

As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Δpmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat. The nucleotide sequence data for wpi6 have been recorded in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AB030210 (cDNA) and AB221353 (genomic DNA).     

DOA1/UFD3 plays a role in sorting ubiquitinated membrane proteins into multivesicular bodies

Ubiquitin (Ub) is a sorting signal that targets integral membrane proteins to the interior of the vacuole/lysosome by directing them into lumenal vesicles of multivesicular bodies (MVBs). The Vps27-Hse1 complex, which is homologous to the Hrs-STAM complex in mammalian cells, serves as a Ub-sorting receptor at the surface of early endosomes. We have found that Hse1 interacts with Doa1/Ufd3. Doa1 is known to interact with Cdc48/p97 and Ub and is required for maintaining Ub levels. We find that the Hse1 Src homology 3 domain binds directly to the central PFU domain of Doa1. Mutations in Doa1 that block Hse1 binding but not Ub binding do not alter Ub levels but do result in the missorting of the MVB cargo GFP-Cps1. Loss of Doa1 also causes a synthetic growth defect when combined with loss of Vps27. Unlike the loss of Doa1 alone, the doa1Delta vps27Delta double mutant phenotype is not suppressed by Ub overexpression, demonstrating that the effect is not due to indirect consequence of lowered Ub levels. Loss of Doa1 results in a defect in the accumulation of GFP-Ub within yeast vacuoles, implying that there is a reduction in the flux of ubiquitinated membrane proteins through the MVB pathway. This defect was also reflected by an inability to properly sort Vph1-GFP-Ub, a modified subunit of the multiprotein vacuolar ATPase complex, which carries an in-frame fusion of Ub as an MVB sorting signal. These results reveal novel roles for Doa1 in helping to process ubiquitinated membrane proteins for sorting into MVBs.     

Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution

Meningococcal and gonococcal outer membrane proteins were reconstituted into liposomes using detergent-mediated dialysis. The detergents octyl glucopyranoside (OGP), sodium cholate and Empigen BB were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. Isopycnic density gradient centrifugation studies showed that liposomes were not formed and hence no protein incorporation occurred during dialysis from an Empigen BB containing reconstitution mixture. Cholate-mediated reconstitution yielded proteoliposomes with only 75% of the protein associated with the vesicles whereas all of the protein was reconstituted into the lipid bilayer during OGP-mediated reconstitution. Essentially complete protein incorporation was achieved with an initial protein-to-lipid ratio of 0.01:1 (w/w) in the reconstitution mixture; however, at higher initial protein-to-lipid ratios (0.02:1) only 75% protein incorporation was achieved. Reconstituted proteoliposomes were observed as large (>300 nm), multilamellar structures using cryo-electron microscopy. Size reduction of these proteoliposomes by extrusion did not result in significant loss of protein or lipid. Extruded proteoliposomes were unilamellar vesicles with mean diameter of about 100 nm.     

Purification and characterization of the S-adenosylmethionine:glutamyl methyltransferase that modifies membrane chemoreceptor proteins in bacteria

The enzyme (EC 2.1.1.24) from Salmonella typhimurium that catalyzes the S-adenosylmethionine-dependent methyl esterification of glutamyl residues in membrane chemoreceptor proteins has been purified to homogeneity, and the nucleotide sequence of the gene coding for this protein, cheR, has been determined. The molecular weight, amino acid composition, and N-terminal amino acid sequence of the purified protein correspond to the values predicted from the sequence of the gene. The pure protein is a 33-kDa monomer. Kinetic studies indicate that, at levels of receptor and S-adenosylmethionine present in wild type cells, the transferase is nearly saturated. The enzyme has a relatively low turnover number, approximately 10 mol of methylester formed per mol of enzyme per min; and there appear to be only approximately 200 methyltransferase monomers per wild type cell.     

Systematic characterization of sperm-specific membrane proteins in swine

To establish a systematic strategy for characterizing fertilization proteins of sperm cells, we prepared alloantisera by immunizing gilts with salt-washed membranes from boar spermatozoa. The antisera recognized a unique subset of sperm membrane proteins that migrated with M(r) 7500-66,000 in SDS-PAGE under nonreducing conditions. The antisera did not recognize proteins of erythrocyte membranes, and tissue absorption experiments further confirmed that the alloantigens were sperm-specific proteins. Each of these sperm-specific membrane proteins (SSMPs) possessed one or more disulfide bonds that were essential for its interaction with alloantibody. Enzymatic deglycosylation revealed that most of the SSMPs were glycoproteins, and their alloantigenicity was not dependent on the presence of N-linked oligosaccharides. The presence of disulfide bonds and glycosylation indicated that the SSMPs identified each comprise at least one extracellular domain. Two-dimensional electrophoresis resolved at least 14 distinct SSMPs, 13 of which possessed acidic pIs (range 4.2-4.8). By indirect immunofluorescence, the SSMPs localized to the cell surface overlying all major regions of the sperm cell. We conclude that the repertoire of immunodominant SSMPs in the pig is relatively small, which makes feasible the systematic elucidation of their functions in fertilization.     

Molecular characterization of mammalian homologues of class C Vps proteins that interact with syntaxin-7

Vesicle-mediated protein sorting plays an important role in segregation of intracellular molecules into distinct organelles. Extensive genetic studies using yeast have identified more than 40 vacuolar protein sorting (VPS) genes involved in vesicle transport to vacuoles. However, their mammalian counterparts are not fully elucidated. In this study, we identified two human homologues of yeast Class C VPS genes, human VPS11 (hVPS11) and human VPS18 (hVPS18). We also characterized the subcellular localization and interactions of the protein products not only from these genes but also from the other mammalian Class C VPS homologue genes, hVPS16 and rVPS33a. The protein products of hVPS11 (hVps11) and hVPS18 (hVps18) were ubiquitously expressed in peripheral tissues, suggesting that they have a fundamental role in cellular function. Indirect immunofluorescence microscopy revealed that the mammalian Class C Vps proteins are predominantly associated with late endosomes/lysosomes. Immunoprecipitation and gel filtration studies showed that the mammalian Class C Vps proteins constitute a large hetero-oligomeric complex that interacts with syntaxin-7. These results indicate that like their yeast counterparts, mammalian Class C Vps proteins mediate vesicle trafficking steps in the endosome/lysosome pathway.     

Molecular characterization of two light-induced, gamete-specific genes from Chlamydomonas reinhardtii that encode hydroxyproline-rich proteins

Gametic differentiation in Chlamydomonas reinhardtii is a two-step process, which is controlled by the sequential action of the two extrinsic signals, nitrogen starvation and blue light. The gamete-specific genes GAS28 and GAS29 are expressed in the late phase of gametogenesis. Their light-induced expression is restricted to cells that have completed the first, nitrogen starvation-activated, phase of differentiation. A comparison of the two genes revealed striking similarities as well as differences. Their most prominent shared feature is an extended sequence homology of over 90% in their 5′-untranslated regions, suggesting a role in translational regulation. GAS28 and GAS29 both encode hydroxyproline-rich proteins (HRGPs) of very similar sizes that exhibit typical features of volvocalean cell wall constituents. GAS28 shows a high degree of homology with the Volvox pherophorin gene family, suggesting a relationship between these genes. Received: 6 August 1998 / Accepted: 16 November 1998     

Evidence that the castor bean allergens are the albumin storage proteins in the protein bodies of castor bean

The well characterized castor bean (Ricinus communis L.) allergens were identified as the low molecular weight albumin storage proteins in the matrix of the protein bodies in the endosperm. The methods of identification involved molecular weight estimation, amino acid composition, stability at 100° C, solubility in various solvents, gel electrophoresis, and immunological techniques. The finding explains the wide distribution of allergens in various seeds.     

Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach

We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.     

Molecular characterization of a heat-modifiable protein from the outer membrane of Escherichia coli.

Discrete fractions of nonhistone chromosomal proteins (NHCP) were obtained from rabbit liver chromatin by their dissociation in 5 m urea with increasing concentrations of NaCl. Three fractions were obtained: M₀, M₁, and M₃. We found that M₀ can modify the conformation of DNA/histone complexes as depicted from their induced increase in the ellipticity of DNA/histone from 5100 to 6900 degree-cm²/dmol. This effect was found to be reversible, while M₁ and M₃ effects, if any, were not measurable. These results suggest that M₀ primarily interacts with the chromatin subunit.     

Isolation and characterization of two cDNA clones that are rapidly induced during the wound response of Arabidopsis thaliana

Differential screening of a cDNA library of Arabidopsis thaliana constructed from the plant tissues harvested 1 h after wounding resulted in isolation of 2 wound-inducible cDNA clones. Kinetic analysis revealed that the corresponding genes are rapidly induced upon wounding. Expression of these clones reached the maximum level around 1–1.5 h after wounding and then were progressively reduced. The time by which expression returned to the control level was around 3 h after wounding. Partial sequence analysis revealed that the two clones are highly homologous to the S-adenosylmethionine synthetase and the glutathione-S-transferase gene, respectively.Abbreviations SAM S-adenosylmethionine - GST glutathione-S-transferase