Cytosolic Protein1

A cytosolic pea (Pisum sativum) seed albumin (ALB) and a chimeric protein (PHALB) consisting of the signal peptide and first three amino acids of phytohemagglutinin (PHA) and the amino acid sequence of ALB were expressed in parallel suspension cultures of tobacco (Nicotiana tabacum) cells and their intracellular fates examined. PHALB was efficiently secreted by the cells whereas ALB remained intracellular. These experiments show that the information contained in the signal peptide of a vacuolar protein is both necessary and sufficient for efficient secretion, and define secretion as a default or bulk-flow pathway. Entry into the secretary pathway was accompanied by glycosylation and the efficient conversion of the high mannose glycans into complex glycans indicating that transported glycoproteins do not need specific recognition domains for the modifying enzymes in the Golgi. Tunicamycin depressed the accumulation of the unglycosylated polypeptide in the culture medium much less than the accumulation of other glycoproteins. We interpret this as evidence that glycans on proteins that are not normally glycosylated do not have the same function of stabilizing and protecting the polypeptide as on natural glycoproteins. The secretary system of plant cells delivers proteins to the vacuole, the tonoplast, the plasma membrane, and the cell wall/extracellular space. In addition, proteins that enter the secretary system may be retained in the endoplasmic reticulum or in various compartments of the Golgi complex. The first step common to the transport of all these proteins is translocation across the ER membrane (25). Once inside the lumen of the ER, transport depends on two types of information: informational domains that contain specific targeting or retention information, and transport competence. Informational domains that result in specific targeting or retention have been identified for yeast and mammalian proteins, but not yet for plant proteins. Transport competence is an as yet poorly defined property that depends on post-translational modifications, solubility, and proper folding of the polypeptide. Proteins that lack transport competence may be broken down in the secretary system (16, 20). In both yeast cells and mammalian cells specific signals are probably not required for secretion. Proteins that have entered the ER and that lack ' Supported by a grant from the National Science Foundation (Cell Biology) and a contract from the United States Department ofEnergy (Basic Energy Biosciences). targeting or retention information are secreted via the bulkflow or default pathway. This is probably also the case in plants. When chimeric constructs of genes encoding various foreign proteins with the nucleotide sequence for a signal peptide were expressed in plant cells, the resulting proteins were secreted. The proteins whose secretion by plant cells has been demonstrated include human serum albumin, a secretory protein (23), GUS,2 PAT, and NPTII, three bacterial cytoplasmic proteins (5), and bacterial chitinase, a secretary protein (18). It is assumed that these mammalian and bacterial proteins were transported nonspecifically from the ER, via the Golgi apparatus to the cell surface. The efficiency of secretion varied as indicated by the retention ofprotein in the secretary system. For example, after 24 h, tobacco cells retained 80% of the GUS activity, 60% of the NPTII activity, and 40% of the PAT activity indicating that the efficiency of secretion depends on the protein that is being transported. To evaluate the information necessary for efficient secretion we examined the intracellular fate of a plant cytosolic protein that is synthesized in transgenic cells as a chimeric protein with the signal peptide of a secretary protein. The chimeric construct that was introduced into tobacco cells consists of the coding sequence for the signal peptide and first three amino acids of PHA, a bean cotyledon vacuolar protein, and the coding sequence of a pea cotyledon cytosolic albumin (12), referred to as ALB. The use of a chimeric gene with the coding sequence for a plant cytosolic protein with cryptic glycosylation sites allows one to create a neoglycoprotein and examine the fate of its glycans. We define a neoglycoprotein here as a protein that is glycosylated although it is not glycosylated in its normal state. We have previously shown that when this chimeric protein called PHALB was expressed in tobacco seeds, the signal peptide of PHA was able to direct its efficient translocation into the ER, where it was glycosylated (6). Subsequently the glycoprotein was transported to the Golgi apparatus, where some of its glycans were modified. In this study we show that PHALB is efficiently secreted by suspension-cultured tobacco cells. The secreted neoglycoprotein contains complex glycans only showing that the conversion ofhigh mannose to complex 2Abbreviations: GUS, ,B-glucuronidase; PAT, phosphinothricin acetyl transferase; NPTII, neomycin phosphotransferase II; ALB, pea seed albumin PA2; PHA, phytohemagglutinin; PHALB, chimeric protein consisting of the first three amino acids ofPHA and the entire sequence of ALB; kb, kilobase; TM, tunicamycin; TFMS, trifluoromethane sulfonic acid; Endo H, endo-#-N-acetylglucosaminidase.