Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae.

Abstract

We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PH05) in multicopy expression plasmids. Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein. Phaseolin polypeptides in S. cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed. We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase. Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S. cerevisiae. Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur. Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation. However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

“…This mechanism has been proposed for the dual localization of the PDI, RB60 (which contains a signal peptide and C-terminal KDEL), to the ER and chloroplast in Chlamydomonas (Levitan et al, 2005). Post-translational insertion of the signal peptide-containing polypeptide was observed in a few cases in animals, yeast and plants (Cramer et al, 1987;Plath et al, 1998;Rapoport et al, 1999). Alternatively, PDI2 could enter the nucleus by interacting with MEE8, which has a strong NLS ( Table 2) that can mask the signal peptide (Karniely and Pines, 2005).…”

The plant vacuolar protein, phytohemagglutinin, is transported to the vacuole of transgenic yeast.

“…Another plant vacuolar storage protein, phaseolin from P, vulgaris, has been expressed in yeast (Cramer et al, 1985(Cramer et al, , 1987. It is glycosylated and remains intracellular.…”

Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco.

“…It has been shown that fava bean lectin, barley hordein, sweet potato sporamin A, tomato polygalacturonase, and wheat high molecular weight glutenin all contain an amino-terminal presequence that can be processed in vitro using canine microsomal membranes (Hemperly et al, 1982;Weber and Brandt, 1985;Hattori et al, 1987;Bulleid and Freedman, 1988;DellaPenna and Bennett, 1988). Furthermore, plant proteins known to be localized in storage bodies or secreted can translocate the ER and trave1 through the secretory pathway of yeast, animal cells, and heterologous plants (Bassüner et al, 1983;Rothstein et al, 1984;Beachy et al, 1985;Greenwood and Chrispeels, 1985;Voelker et al, 1986;Cramer et al, 1987;Hoffman et al, 1987;Neill et al, 1987;Tague and Chrispeels, 1987;Bustos et al, 1988;Sturm et al, 1988;Wallace et al, 1988).…”

“…It has been shown that fava bean lectin, barley hordein, sweet potato sporamin A, tomato polygalacturonase, and wheat high molecular weight glutenin all contain an amino-terminal presequence that can be processed in vitro using canine microsomal membranes (Hemperly et al, 1982; Weber and Brandt, 1985;Hattori et al, 1987;Bulleid and Freedman, 1988;DellaPenna and Bennett, 1988). Furthermore, plant proteins known to be localized in storage bodies or secreted can translocate the ER and trave1 through the secretory pathway of yeast, animal cells, and heterologous plants (Bassüner et al, 1983;Rothstein et al, 1984;Beachy et al, 1985; Greenwood and Chrispeels, 1985;Voelker et al, 1986;Cramer et al, 1987; Hoffman et al, 1987;Neill et al, 1987;Tague and Chrispeels, 1987; Bustos et al, 1988; Sturm et al, 1988; Wallace et al, 1988).A useful way of, studying the function of these signal sequences, and defining their important domains, is to construct hybrid proteins containing the putative signal peptide and a protein that is normally located in a different compartment and to study their translocation in vitro using microsomes (Blobel and Dobberstein, 1975) and in vivo by introducing genes encoding the hybrid proteins into plants using transformation. In the latter case, studies have been conducted using the transit peptides of the small subunit of ribulose-l,5-bisphophate carboxylase/oxygenase fused to neomycin phosphotransferase (Schreier et al, 1985;Van den Broeck et al, 1985), chlorophyll a/b binding protein fused to P-glucuronidase (GUS) (Kavanagh et al, 1988) and the p subunit of ATPase fused to chloramphenicol acetyltransferase (CAT) (Boutry et al, 1987).…”