Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme

The role of structural signals in intercompartmental transport has been addressed by the isolation of yeast invertase (SUC2) mutations that cause intracellular accumulation of active enzyme. Two mutations that delay transport of core-glycosylated invertase, but not acid phosphatase, have been mapped in the 5' coding region of SUC2. Both mutations reduce specifically the transport of invertase to a compartment, presumably in the Golgi body, where outer chain carbohydrate is added. Subsequent transport to the cell surface is not similarly delayed. One mutation (SUC2-s1) converts an ala codon to val at position -1 in the signal peptide; the other (SUC2-s2) changes a thr to an ile at position +64 in the mature protein. Mutation s1 results in about a 50-fold reduced rate of invertase transport to the Golgi body which is attributable to defective signal peptide cleavage. While peptide cleavage normally occurs at an ala-ser bond, the s1 mutant form is processed slowly at the adjacent ser-met position giving rise to mature invertase with an N-terminal met residue. s2 mutant invertase is transported about sevenfold more slowly than normal, with no delay in signal peptide cleavage, and no detectable abnormal physical property of the enzyme. This substitution may interfere with the interaction of invertase and a receptor that facilitates transport to the Golgi body

The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information

The NH2 terminus of the yeast F1-ATPase beta subunit precursor directs the import of this protein into mitochondria. To define the functionally important components of this import signal, oligonucleotide-directed mutagenesis was used to introduce a series of deletion and missense mutations into the gene encoding the F1-beta subunit precursor. Among these mutations were three nonoverlapping deletions, two within the 19-amino-acid presequence (delta 5-12 and delta 16-19) and one within the mature protein (delta 28-34). Characterization of the mitochondrial import properties of various mutant F1-beta subunit proteins containing different combinations of these deletions showed that import was blocked only when all three deletions were combined. Mutant proteins containing all possible single and pairwise combinations of these deletions were found to retain the ability to direct mitochondrial import of the F1-beta subunit. These data suggest that the F1-beta subunit contains redundant import information at its NH2 terminus. In fact, we found that deletion of the entire F1-beta subunit presequence did not prevent import, indicating that a functional mitochondrial import signal is present near the NH2 terminus of the mature protein. Furthermore, by analyzing mitochondrial import of the various mutant proteins in [rho-] yeast, we obtained evidence that different segments of the F1-beta subunit import signal may act in an additive or cooperative manner to optimize the import properties of this protein

Influence of Different Concentrations of Salt Stress on In Vitro Multiplication of Some Fig (Ficus carcia L.) Cultivars

This work aimed to establish in-vitro plant formation from Ficus carica L. cultivars (i.e., Black Mission, Brown Turkey and Brunswick) and investigate their salt tolerance using different concentrations of NaCl. Plant growth regulators BAP, Kinetin, 2iP and NAA were evaluated during proliferation, elongation and rooting stages. Murashige and Skoog (MS) was the most superior medium for increasing explant development in most tissue culture techniques for all cultivars. BA was the best cytokinin for enhancing proliferation in cvs. Brown Turkey and Brunswick, while Kinetin was more effective in improving growth and greening parameters of cv. Black Mission. Shoot elongation of cv. Black Mission was increased by using hormone-free media. Shoot elongation was enhanced by addition of 0.5 mg L-1 GA3 to the culture medium of cv. Brunswick, while 1.0 mg L-1 was better for cv. Brown Turkey. IBA at 2.0 mg L-1 was most effective in maximizing cv. Brown Turkey rooting, while 1.5 mg L-1 was the best for the other cultivars. Half strength MS medium produced the best root formation compared to other media strengths. NaCl at concentrations of more than 12 g L-1 induced lethal effects on all parameters under study and 11 g L-1 had adverse effect on the plantlets of cv. Brunswick. Contents of K+, Na +, Fe++ and Zn++ increased incrementally with increasing NaCl levels in all cultivars. cv. Brown Turkey accumulated more K+ and Na+ than others

The Saccharomyces cerevisiae SEC14 Gene Encodes a Cytosolic Factor That Is Required for Transport of Secretory Proteins from the Yeast Golgi Complex

We have obtained and characterized a genomic clone of SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex. Gene disruption experiments indicated that SEC14 is an essential gene for yeast vegetative growth. Nucleotide sequence analysis revealed the presence of an intron within the SEC14 structural gene, and predicted the synthesis of a hydrophilic polypeptide of 35 kD in molecular mass. In confirmation, immunoprecipitation experiments demonstrated SEC14p to be an unglycosylated polypeptide, with an apparent molecular mass of some 37 kD, that behaved predominantly as a cytosolic protein in subcellular fractionation experiments. These data were consistent with the notion that SEC14p is a cytosolic factor that promotes protein export from yeast Golgi. Additional radiolabeling experiments also revealed the presence of SEC14p-related polypeptides in extracts prepared from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. Furthermore, the K. lactis SEC14p was able to functionally complement S. cerevisiae sec14ts defects. These data suggested a degree of conservation of SEC14p structure and function in these yeasts species

The Amino Terminus of the Yeast F_1-ATPase β-Subunit Precursor Functions as a Mitochondrial Import Signal

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells

Impact of application of zinc oxide nanoparticles on callus induction, plant regeneration, element content and antioxidant enzyme activity in tomato (Solanum lycopersicum MILL) under salt stress

The properties of nanomaterials and their potential applications have been given considerable attention by researchers in various fields, especially agricultural biotechnology. However, not much has been done to evaluate the role or effect of zinc oxide nanoparticles (ZnO-NP) in regulating physiological and biochemical processes in response to salt-induced stress. For this purpose, some callus growth traits, plant regeneration rate, mineral element (sodium, potassium, phosphorous and nitrogen) contents and changes in the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in tissues of five tomato cultivars were investigated in a callus culture exposed to elevated concentrations of salt (3.0 and 6.0 g L-1NaCl), and in the presence of zinc oxide nanoparticles (15 and 30 mg L-1). The relative callus growth rate was inhibited by 3.0 g L-1 NaCl; this was increased dramatically at 6.0 g L-1. Increasing exposure to NaCl was associated with a significantly higher sodium content and SOD and GPX activities. Zinc oxide nanoparticles mitigated the effects of NaCl, and in this application of lower concentrations (15 mg L-1) was more effective than a higher concentration (30 mg L-1). This finding indicates that zinc oxide nanoparticles should be investigated further as a potential anti-stress agent in crop production. Different tomato cultivars showed different degrees of tolerance to salinity in the presence of ZnO-NP. The cultivars Edkawy, followed by Sandpoint, were less affected by salt stress than the cultivar Anna Aasa