The plant vacuolar sorting receptor AtELP is involved in transport of NH(2)-terminal propeptide-containing vacuolar proteins in Arabidopsis thaliana

Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH(2)-terminal propeptide (NTPP) present in sweet potato sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barley aleurain and Spo, but not with the CTPPs of BL or tobacco chitinase, in a pH-dependent and sequence-specific manner. In addition, EM studies revealed the colocalization of AtELP with NTPP-Spo at the Golgi apparatus, but not with BL-CTPP in roots of transgenic Arabidopsis plants. Further, we found that AtELP interacts in a similar manner with the NTPP of the endogenous vacuolar protein AtALEU (Arabidopsis thaliana Aleu), a protein highly homologous to barley aleurain. We hypothesize that AtELP functions as a vacuolar sorting receptor involved in the targeting of NTPP-, but not CTPP-containing proteins in Arabidopsis.     

The N-terminal propeptide and the C terminus of the precursor to 20-kilo-dalton potato tuber protein can function as different types of vacuolar sorting signals

Two types of vacuolar sorting signals (VSSs), an asparagine-proline-isoleucine-arginine-leucine (NPIRL)-related VSS in the N-terminal propeptides (NTPPs) and a C-terminal VSS in the C-terminal propeptides (CTPPs), function differently in plant cells. A precursor to a 20-kDa protein of potato tuber (PT20) contains two NPIRL-related sequences, NPINL in a short NTPP and NPLDV close to the C terminus of the precursor. We made mutant forms of sweet potato sporamin (SPO), nPT20-SPO, in which the N-terminal pre-pro part was exchanged with that of the precursor to PT20, and SPO-PT20c, in which the C-terminal 13 amino acids of the precursor to PT20 was attached to the C terminus of delta pro-SPO which lacked NTPP. Both nPT20-SPO and SPO-PT20c were efficiently transported to the vacuoles in tobacco cells. Unlike nPT20-SPO, the vacuolar transport of SPO-PT20c was inhibited by wortmannin and by the C-terminal addition of Gly or Gly-Gly suggesting its similarity to the vacuolar transport of sporamin mediated by CTPP of barley lectin. Further analysis of the C-terminal sequence of PT20 indicated that the most C-terminal SFKQVQ sequence functions as the C-terminal VSS. These results suggest that the precursor to PT20 contains both NPIRL-like VSS in its NTPP and C-terminal VSS at the C terminus.     

A carboxyl-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco.

Barley lectin is synthesized as a preproprotein with a glycosylated carboxyl-terminal propeptide (CTPP) that is removed before or concomitant with deposition of the mature protein in vacuoles. Expression of a cDNA clone encoding barley lectin in transformed tobacco plants results in the correct processing, maturation, and accumulation of active barley lectin in vacuoles [Wilkins, T.A., Bednarek, S.Y., and Raikhel, N.V. (1990). Plant Cell 2, 301-313]. The glycan of the propeptide is not essential for vacuolar sorting, but may influence the rate of post-translational processing [Wilkins, T.A., Bednarek, S.Y., and Raikhel, N.V. (1990). Plant Cell 2, 301-313]. To investigate the functional role of the CTPP in processing, assembly, and sorting of barley lectin to vacuoles, a mutant barley lectin cDNA clone lacking the 15-amino acid CTPP was prepared. The CTPP deletion mutant of barley lectin was expressed in tobacco protoplasts, suspension-cultured cells, and transgenic plants. In all three systems, the wild-type barley lectin was sorted to vacuoles, whereas the mutant barley lectin was secreted to the incubation media. Therefore, we conclude that the carboxyl-terminal domain of the barley lectin proprotein is necessary for the efficient sorting of this protein to plant cell vacuoles.     

Post-translational maturation of natural and drug-induced missorted phytohemagglutinin

The bean lectin phytohemagglutinin (PHA) was expressed in transgenic suspension-cultured BY-2 tobacco cells simultaneously with another recombinant vacuolar protein, the sweet potato sporamin. In contrast to previous observations in different transgenic plant systems when expressed in BY-2 tobacco cells, phytohemagglutinin is mostly but not exclusively targeted to the vacuole. Indeed, a small amount of recombinant phytohemagglutinin is secreted into the culture medium of tobacco cells. Furthermore part of this extracellular phytohemagglutinin has no lectin activity and presents an abnormal glycosylation consistent with higher accessibility of glycans N-linked to these extracellular phytohemagglutinin forms. Phytohemagglutinin secretion occurs regardless of recombinant protein expression level. Consequently, missorting in this case is due to an abnormal phytohemagglutinin conformation or oligomerization rather than to receptor saturation. The treatment of BY-2 cells with drugs, such as monensin and wortmannin, increases even more the transport of phytohemagglutinin to the cell surface through a general inhibition of the sorting mechanisms of vacuolar proteins. The sensitivity to wortmannin is similar for the sorting of phytohemagglutinin and endogenous tobacco chitinase and β-1,3-glucanase, suggesting that phytohemagglutinin and COOH-terminal propeptide mediated vacuolar sorting share similar mechanisms. A characterization of glycans N-linked to extracellular phytohemagglutinin secreted by monensin- or wortmannin-treated transgenic tobacco cells illustrates that in contrast with monensin, wortmannin completely inhibits the sorting of vacuolar proteins without having any effect on the efficiency of Golgi processing enzymes.     

Colocalization of barley lectin and sporamin in vacuoles of transgenic tobacco plants.

Various targeting motifs have been identified for plant proteins delivered to the vacuole. For barley (Hordeum vulgare) lectin, a typical Gramineae lectin and defense-related protein, the vacuolar information is contained in a carboxyl-terminal propeptide. In contrast, the vacuolar targeting information of sporamin, a storage protein from the tuberous roots of the sweet potato (Ipomoea batatas), is encoded in an amino-terminal propeptide. Both proteins were expressed simultaneously in transgenic tobacco plants to enable analysis of their posttranslational processing and subcellular localization by pulse-chase labeling and electron-microscopic immunocytochemical methods. The pulse-chase experiments demonstrated that processing and delivery to the vacuole are not impaired by the simultaneous expression of barley lectin and sporamin. Both proteins were targeted quantitatively to the vacuole, indicating that the carboxyl-terminal and amino-terminal propeptides are equally recognized by the vacuolar protein-sorting machinery. Double-labeling experiments showed that barley lectin and sporamin accumulate in the same vacuole of transgenic tobacco (Nicotiana tabacum) leaf and root cells.     

Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and <Emphasis Type="Italic">Arabidopsis</Emphasis>

Plant vacuoles are multi-functional, developmentally varied and can occupy up to 90% of plant cells. The N-terminal propeptide (NTPP) of sweet potato sporamin and the C-terminal propeptide (CTPP) of tobacco chitinase have been developed as models to target some heterologous proteins to vacuoles but so far tested on only a few plant species, vacuole types and payload proteins. Most studies have focused on lytic and protein-storage vacuoles, which may differ substantially from the sugar-storage vacuoles in crops like sugarcane. Our results extend the evidence that NTPP of sporamin can direct heterologous proteins to vacuoles in diverse plant species and indicate that sugarcane sucrose-storage vacuoles (like the lytic vacuoles in other plant species) are hostile to heterologous proteins. A low level of cytosolic NTPP-GFP (green fluorescent protein) was detectable in most cell types in sugarcane and Arabidopsis, but only Arabidopsis mature leaf mesophyll cells accumulated NTPP-GFP to detectable levels in vacuoles. Unexpectedly, efficient developmental mis-trafficking of NTPP-GFP to chloroplasts was found in young leaf mesophyll cells of both species. Vacuolar targeting by tobacco chitinase CTPP was inefficient in sugarcane, leaving substantial cytoplasmic activity of rat lysosomal -glucuronidase (GUS) [ER (endoplasmic reticulum)-RGUS-CTPP]. Sporamin NTPP is a promising targeting signal for studies of vacuolar function and for metabolic engineering. Such applications must take account of the efficient developmental mis-targeting by the signal and the instability of most introduced proteins, even in storage vacuoles.     

The N-terminal propeptide of the precursor to sporamin acts as a vacuole-targeting signal even at the C terminus of the mature part in tobacco cells.

An asparagine-proline-isoleucine-arginine-leucine (NPIRL) and its related sequences in the N-terminal propeptides (NTPP) of several plant vacuolar proteins, including that of sporamin from sweet potato (SPO) function as vacuole-targeting determinants in a manner that is distinct from the vacuole-targeting determinant in the CTPPs of other plant vacuolar proteins. When the mutant precursor to sporamin, SPO-NTPP (in which NTPP was moved to the C terminus of the mature part), was expressed in tobacco (Nicotiana tabacum) cells, the pro-form was efficiently targeted to the vacuole and the NTPP was cleaved off. Unlike the results obtained with the wild-type precursor, substitution of the NPIRL sequence in the C-terminally located NTPP to asparagine-proline-glycine-arginine-leucine in the SPO-isoleucine-28-to-glycine mutant resulted in missorting of less than 20% of the pro-form to the medium. Unlike the vacuolar transport of SPO-NTPP, the vacuolar transport of SPO-isoleucine-28-to-glycine was strongly inhibited by 33 microM wortmannin, which is similar to the C-terminal propeptide-mediated vacuolar transport. These results suggest that the vacuole-targeting function of the NPIRL sequence is not strictly dependent on its location at the N terminus of a protein and that the C-terminally located mutant NTPP acquired some physicochemical properties of the C-terminal vacuole-targeting sequence.     

Cloning and sequence analysis of the ces10 gene encoding a Sphingomonas paucimobilis esterase

Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals; and, in plants, they have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein is shown to be synthesized as a preprotein with both a N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, using an analytical system of transgenically cultured tobacco cells (Nicotiana tabacum, BY2). Here, we report that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. We also demonstrate that HRP C1a derived from a preprotein lacking CTPP is shunted into the secretory pathway.     

A carboxy-terminal plant vacuolar targeting signal is not recognized by yeast

Three different classes of signals for plant vacuolar targeting have been defined. Previous work has demonstrated that the carboxyl-terminal propeptide (CTPP) of barley lectin (BL) is a vacuolar targeting signal in tobacco plants. When a mutant BL protein lacking the CTPP is expressed in tobacco, the protein is secreted. In an effort to determine the universality of this signal, the CTPP was tested for its ability to target proteins to the vacuole of Saccharomyces cerevisiae. Genes encoding fusion proteins between the yeast secreted protein invertase and BL domains were synthesized and transformed into an invertase deletion mutant of yeast. Invertase assays on intact and detergent-solubilized cells demonstrated that invertase+CTPP was secreted, while nearly 90% of the invertase::BL+CTPP (fusion protein between invertase and BL containing the CTPP) and invertase::BL-CTPP proteins (fusion between invertase and BL lacking the CTPP) were retained intracellularly. These fusions were secreted in a mutant of yeast that normally secretes proteins targeted to the vacuole. With this and previous work, proteins representing all three classes of plant vacuolar targeting signals have now been tested in yeast, and in all cases, the experiments indicate that the plant proteins are directed to the yeast vacuole using signals other than those recognized by plants.     

The barley lectin carboxyl-terminal propeptide is a vacuolar protein sorting determinant in plants.

We have previously shown that the 15-amino acid carboxyl-terminal propeptide of probarley lectin is necessary for the proper sorting of this protein to the plant vacuole. A mutant form of the protein lacking the carboxyl-terminal propeptide is secreted. To test whether the carboxyl-terminal propeptide is the vacuole sorting determinant of probarley lectin, we examined in transgenic tobacco the processing and sorting of a series of fusion proteins containing the secreted protein, cucumber chitinase, and regions of probarley lectin. Pulse-labeling experiments demonstrated that the fusion proteins were properly translocated through the tobacco secretory system and that cucumber chitinase and cucumber chitinase fusion proteins lacking the carboxyl-terminal propeptide were secreted. The cucumber chitinase fusion protein containing the carboxyl-terminal propeptide was properly processed and sorted to the vacuole in transgenic tobacco as confirmed by organelle fractionation and electron microscopy immunocytochemistry. Therefore, the barley lectin carboxyl-terminal propeptide is both necessary and sufficient for protein sorting to the plant vacuole.     

The internal propeptide of the ricin precursor carries a sequence-specific determinant for vacuolar sorting

Ricin is a heterodimeric toxin that accumulates in the storage vacuoles of castor bean (Ricinus communis) endosperm. Proricin is synthesized as a single polypeptide precursor comprising the catalytic A chain and the Gal-binding B chain joined by a 12-amino acid linker propeptide. Upon arrival in the vacuole, the linker is removed. Here, we replicate these events in transfected tobacco (Nicotiana tabacum) leaf protoplasts. We show that the internal linker propeptide is responsible for vacuolar sorting and is sufficient to redirect the ricin heterodimer to the vacuole when fused to the A or the B chain. This internal peptide can also target two different secretory protein reporters to the vacuole. Moreover, mutation of the isoleucine residue within an NPIR-like motif of the propeptide affects vacuolar sorting in proricin and in the reconstituted A-B heterodimer. This is the first reported example of a sequence-specific vacuolar sorting signal located within an internal propeptide.     

Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualized by green fluorescent proteins targeted to either type of vacuoles

Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two different vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-neutral vacuole by the C-terminal vacuolar sorting determinant of tobacco chitinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-specific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuolated (mini-) protoplasts regenerate a large CV. Expression of the two vacuolar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic vacuole, whereas neutral vacuoles always remained peripheral. Only later, once the regeneration of the CV was completed, the content of peripheral storage vacuoles could be seen to appear in the CV of a third of the cells, apparently by heterotypic fusion.     

Targeting of proConA to the plant vacuole depends on its nine amino-acid C-terminal propeptide

Concanavalin A (ConA) is a well characterized and extensively used lectin accumulated in the protein bodies of jack bean cotyledons. ConA is synthesized as an inactive precursor proConA. The maturation of inactive proConA into biologically active ConA is a complex process including the removal of an internal glycopeptide and a C-terminal propeptide (CTPP), followed by a head-to-tail ligation of the two largest polypeptides. The cDNA encoding proConA was cloned and expressed in tobacco BY-2 cells. ProConA was slowly transported to the vacuole where its maturation into ConA was similar to that in jack bean cotyledons, apart from an incomplete final ligation. To investigate the role of the nine amino acid CTPP, a truncated form lacking the propeptide (proConADelta9) was expressed in BY-2 cells. In contrast to proConA, proConADelta9 was rapidly chased out of the endoplasmic reticulum (ER) and secreted into the culture medium. The CTPP was then fused to the C-terminal end of a secreted form of green fluorescent protein (secGFP). When expressed in tobacco BY-2 cells and leaf protoplasts, the chimaeric protein was located in the vacuole whereas secGFP was located in the culture medium and in the vacuole. Altogether, our results show we have isolated a new C-terminal vacuolar sorting determinant.     

Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants

The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and -1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.     

Transport of a Sweet Potato Storage Protein, Sporamin, to the Vacuole in Yeast Cells

The propeptide of a precursor to sporamin, a storage proteinof sweet potato, is required for targeting of sporamin to thevacuole in transformed tobacco cells (Matsuoka and Nakamura1991). A fusion gene consisting of an inducible GAL 10 promoterand sporamin cDNA was introduced into Saccharomyces cerevisiaeby use either of a multiple-copy plasmid (YEpSAD16) or of asingle-copy plasmid (YCpSAD16) to control the level of expressionof the precursor. Although we could not detect any sporamin-relatedpolypeptides in cells that harbored YCpSAD16, extracts fromcells that harbored YEpSAD16 contained multiple forms of sporaminrelatedpolypeptides: preprosporamin, prosporamin and several polypeptidesthat were smaller than prosporamin. However, YCpSAD16 directedthe accumulation of prosporamin in pep4 mutant yeast cells thatlack vacuolar proteases, andpep4 mutant cells that harboredYEpSAD16 did not contain any sporamin-related polypeptides smallerthan prosporamin. The vacuole fractions isolated from the wild-typeand pep4 mutant cells contained sporamin-related polypeptidessmaller than prosporamin and prosporamin, respectively. Theseand other results suggest that, at a low level of expressionof the precursor, prosporamin is transported to the vacuoleand degraded by vacuolar proteases. A mutant precursor to sporamin,in which the propeptide and the N-terminal region of maturesporamin were replaced by an unrelated sequence of four aminoacid residues, directed the secretion of sporamin to the culturemedium in transformed tobacco cells. However, this mutationdid not affect the transport of sporamin to the vacuole in yeastcells and none of the sporamin-related polypeptides were secretedto the extracellular space. (Received July 16, 1991; Accepted March 25, 1992)     

Determination of the functional elements within the vacuolar targeting signal of barley lectin.

We have previously demonstrated that the carboxyl-terminal propeptide of barley lectin is both necessary and sufficient for protein sorting to the plant vacuole. Specific mutations were constructed to determine which amino acid residues or secondary structural determinants of the carboxyl-terminal propeptide affect proper protein sorting. We have found that no consensus sequence or common structural determinants are required for proper sorting of barley lectin to the vacuole. However, our analysis demonstrated the importance of hydrophobic residues in vacuolar targeting. In addition, at least three exposed amino acid residues are necessary for efficient sorting. Sorting was disrupted by the addition of two glycine residues at the carboxyl-terminal end of the targeting signal or by the translocation of the glycan to the carboxy terminus of the propeptide. These results suggest that some components of the sorting apparatus interact with the carboxy terminus of the propeptide.     

Interaction of a potential vacuolar targeting receptor with amino- and carboxyl-terminal targeting determinants.

A protein of 80 kD from developing pea (Pisum sativum) cotyledons has previously been shown to exhibit characteristics of a vacuolar targeting receptor by means of its affinity for the amino-terminal vacuolar targeting sequence of proaleurain from barley (Hordeum vulgare). In this report we show that the same protein also binds to the amino-terminal targeting peptide of prosporamin from sweet potato (Ipomoea batatas) and to the carboxyl-terminal targeting determinant of pro-2S albumin from Brazil nut (Bertholletia excelsa). The receptor protein does not bind to the carboxyl-terminal propeptide (representing the targeting sequence) of barley lectin. The binding of the 80-kD protein to the sporamin determinant involves a motif (NPIR) that has been shown to be crucial for vacuolar targeting in vivo. The binding to the carboxyl-terminal targeting determinant of pro-2S albumin appears to involve the carboxyl-terminal propeptide and the adjacent five amino acids of the mature protein. The 80-kD protein does not bind to peptide sequences that have been shown to be incompetent in directing vacuolar targeting.     

Large alkyl side-chains of isoleucine and leucine in the NPIRL region constitute the core of the vacuolar sorting determinant of sporamin precursor

The N-terminal propeptide of the sporamin precursor contains vacuolar targeting information within the Asn-26/Pro-27/Ile-28/Arg-29/Leu-30 (NPIRL) sequence. An Agrobacterium-mediated transient expression assay with tobacco BY-2 cells was employed to investigate the role of each amino acid of the NPIRL region in vacuolar targeting. Replacement of Asn-26, Pro-27, Ile-28 and Leu-30 with several amino acids caused secretion of the mutant prosporamin. Leu was the only amino acid that could be substituted for Ile-28 without affecting transport. Exchange of Leu-30 for amino acids with small side-chains abolished vacuolar delivery. These results indicate that the consensus composition of the NPIRL sequence is [preferably Asn]-[not acidic]-[Ile or Leu]-[any amino acid]-[large and hydrophobic] and suggest that the large alkyl side-chains of Ile-28 and Leu-30 constitute the core of the vacuolar sorting determinant.     

The phaseolin vacuolar sorting signal promotes transient, strong membrane association and aggregation of the bean storage protein in transgenic tobacco

Vacuolar storage proteins of the 7S class are co-translationally introduced into the endoplasmic reticulum and reach storage vacuoles via the Golgi complex and dense vesicles. The signal for vacuolar sorting of one of these proteins, phaseolin of Phaseolus vulgaris, consists of a four-amino acid hydrophobic propeptide at the C-terminus. When this sequence is deleted, phaseolin is secreted instead of being sorted to vacuoles. It is shown here that in transgenic tobacco plants newly-synthesized phaseolin has unusual affinity to membranes and forms SDS-resistant aggregates, but mutated phaseolin polypeptides that are either secreted or defective in assembly do not have these characteristics. Association to membranes and aggregation are transient events: phaseolin accumulated in vacuoles is soluble in the absence of detergents and is not aggregated. Association to membranes starts before the phaseolin glycan acquires a complex structure and therefore before the protein reaches the medial or trans-cisternae of the Golgi complex. These results support the hypothesis of a relationship between aggregation and vacuolar sorting of phaseolin and indicate that sorting may start in early compartments of the secretory pathway.     

O-glycosylation of a precursor to a sweet potato vacuolar protein, sporamin, expressed in tobacco cells

Sporamin, a vacuolar protein of the sweet potato, is synthesized as a precursor that contains signal peptide and an N-terminal propeptide that functions as a vacuolar targeting determinant. Sporamin, when expressed in tobacco cells, migrated as smeared bands on an SDS-polyacrylamide gel. The smearing was due to O-glycosylation of the precursor to sporamin. The smeared bands were stained by a glycan-specific stain but no N-glycosylation site was found in the amino acid sequence of the precursor to sporamin. The glycan attached to sporamin contained galactose and arabinose as major sugar components. Mutations that altered the Pro³⁶ or Ser³⁹ residue of the precursor to sporamin prevented glycosylation of the protein, and analysis by semiquantitative Edman degradation suggested that a glycan moiety was attached to Pro³⁶ and, possibly, to Ser³⁹. Pulse-labeling and cell-fractionation experiments revealed that the O-glycosylation of the precursor to sporamin occurred in the Golgi apparatus. Thus, this modification serves as a good marker of the transport from the endoplasmic reticulum (ER) to the Golgi apparatus of the precursor to sporamin. Treatment of transformed tobacco cells with brefeldin A (BFA) caused the intracellular accumulation of prosporamin that did not migrate as smeared bands. Thus, it appeared that BFA inhibited the transport of the precursor to sporamin to the Golgi apparatus. This result provides the first biochemical evidence that BFA inhibits transport from the ER to the Golgi apparatus in plant cells.