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The Plant Cell, Vol. 9, 1869-1880, October 1997 O 1997 American Society of Plant Physiologists Protein Quality Control along the Route to the Plant Vacuole Emanuela Pedrazzini,a,l Giovanna Giovinazzo,a,2Anna Bielli,a Maddalena de Virgilio,a Lorenzo Frigerio,a,b Míchela Pesca,a F r a n c o Faoro,c Roberto Bollini,aAldo Ceriotti,a and A l e s s a n d r o Vitaleas3 alstituto Biosintesi Vegetali, Consiglio Nazionale delle Ricerche, via Bassini 15, 201 33 Milan, ltaly bDepartment of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom =Centro Miglioramento Sanitario Colture Agrarie, Consiglio Nazionale delle Ricerche, via Celoria 2, 201 33 Milan, ltaly To acquire information on the relationships between structural maturation of proteins in the endoplasmic reticulum (ER) and their transport along the secretory pathway, we have analyzed the destiny of an assembly-defective form of the trimeric vacuolar storage glycoprotein phaseolin. In leaves of transgenic tobacco, where assembly-competent phaseolin is correctly targeted t o the vacuole, defective phaseolin remains located in the ER or a closely related compartment where it represents a major ligand of the chaperone BiP. Defective phaseolin maintained susceptibility to endoglycosidase H and was slowly degraded by a process that is not inhibited by heat shock or brefeldin A, indicating that degradation does not involve transport along the secretory pathway. These results provide evidence for the presente of a quality control mechanism in the ER of plant cells that avoids intracellular trafficking of severely defective proteins and eventually leads t o their degradation. INTRODUCTION Vacuolar storage proteins reach their oligomeric structure mainly before they exit from the endoplasmic reticulum (ER), where they have been cotranslationally inserted to be transported via the Golgi complex to their destination along the secretory pathway (Shewry et al., 1995; Vitale and Bollini, 1995). Quaternary structure is the major determinant of the resistance of 7 s and 11s storage proteins to proteolysis (Deshpande and Damodaran, 1989; Dickinson et al., 1989; Ceriotti et al., 1991). Such resistance is probably necessary for their efficient accumulation in protein storage vacuoles: in fact, these compartments also accumulate proteolytic enzymes, although their hydrolytic activity is lower than that of vacuoles of vegetative tissues (Okita and Rogers, 1996). In this study, we used a storage protein to investigate whether plant cells possess a mechanism that senses the leve1 of structural maturation of newly synthesized proteins and allows their intracellular traffic only upon acquisition of their correct conformation. There is much evidence that such a mechanism operates during the synthesis of many oligomeric proteins in mammalian cells and of at least two vacuolar proteins in yeast cells (Finger et al., 1993; Hammond and Helenius, 1995).The details of the molecular interactions that allow this process to occur are not yet fully understood; ’ Current address: Centro di Farmacologia Cellulare e Molecolare, Consiglio Nazionale delle Ricerche, via Vanvitelli 32, 20129 Milan, Italy. * Current address: lstituto di Ricerca sulle Biotecnologie Agroalimen- tari, Consiglio Nazionale delle Ricerche, via prov. Lecce-Monteroni, 731O0 Lecce, Italy. To whom correspondence should be addressed. E-mail vitale@ icm.mi.cnr.it; fax 39-2-23699411. however, interactions of newly synthesized polypeptides with ER resident proteins, whose roles are to assist correct structural maturation, seem to be important in the process of ER retention. Proteins that fail to reach the correct maturation because of permanent structural defects are eventually degraded by the system, in some cases via retrotranslocation from the ER into the cytosol (Lord, 1996).Therefore, the system described for mammalian and yeast cells, termed quality control, has two functions, retention and degradation. The major storage protein of common bean, phaseolin, is a homotrimeric protein of the 7s class (Lawrence et al., 1994). We have shown that one of the ER residents, the binding protein (BiP), is found in association with phaseolin before this assembles into trimers in the ER (Vitale et al., 1995). BiP is a molecular chaperone of the heat shock 70 class and assists protein maturation in the ER, probably by inhibiting hydrophobic interactions that may lead to permanent misfolding of folding and assembly intermediates (Pedrazzini and Vitale, 1996).We have also found that an assembly-defective form of phaseolin, in which a C-terminal domain involved in assembly has been deleted, shows more extensive association with BiP than does wild-type PHSL, when transiently expressed in tobacco protoplasts (Pedrazzini et al., 1994).This truncated phaseolin is impaired in intracellular transport along the secretory pathway when expressed in Xenopus oocytes, indicating that it is subjected to quality control in animal cells (Ceriotti et al., 1991). We show here that this defective phaseolin expressed in transgenic tobacco remains confined to the ER or a closely related compartment and is degraded independently of the Golgi 1870 The Plant Cell complex-mediated route of protein transport to the vacuole. These results provide direct evidence for quality control in the plant secretory pathway. RESULTS Assembly-Defective Phaseolin Is Located in the ER or a Closely Related Compartment The assembly-defective A363 phaseolin, schematically represented in Figure 1, was subcloned into plasmid pDHA to allow constitutive expression driven by the cauliflower mosaic virus 35S promoter (Pedrazzini et al., 1994; Tabe et al., 1995) and was stably expressed in tobacco by Agrobacterium-mediated transformation. We previously determined that A363 expressed transiently in tobacco mesophyll protoplasts is correctly translocated into the ER lumen and glycosylated but remains monomeric and shows extensive interaction with BiP before being degraded (Pedrazzini et al., 1994). We first determined the banding pattern, level of accumulation, and intracellular location of A363 in leaves of transgenic tobacco; in subsequent experiments, leaves were used as a source of protoplasts for pulse-chase analysis of the stability and intracellular traffic of the defective phaseolin. Leaves were homogenized in the presence of nonionic detergent, and the solubilized proteins were separated by SDS-PAGE and immunodetected on gel blots by use of an anti-phaseolin antiserum. As shown in Figure 2, no signal was detectable in leaf extracts from transgenic plants transformed with plasmid pDHA without inserts (Figure 2, lane 7). A363 accumulated as a single polypeptide with an M, of 46 kD (lanes 8 and 9, two independent transformants). This is the size expected for fully glycosylated A363 phaseolin (Pedrazzini et al., 1994; phaseolin contains two N-glycosylation sites, Asn-252 and Asn-341). Extraction of phaseolin in the presence of nonionic detergent was quantitative, be- wild-type A363 T343FA360 L_L V//////////M FT Figure 1. Schematic Representation of Phaseolin Polypeptides Encoded by the DMA Constructs Used in This Work. The signal peptide for insertion into the ER (stippled rectangles), the p-barrel domains (hatched rectangles), the a-helical domains involved in assembly (black rectangles), and the glycosylation sites (M') are highlighted. White rectangles represent interconnecting and terminal regions. 1 2 3 4 5 6 7 8 9 Figure 2. Accumulation of Phaseolin Constructs in Leaves of Transgenic Tobacco. Proteins were detected by SDS-PAGE and protein gel blot analysis using the anti-phaseolin antiserum. Lanes 1 to 3 contain 1 ^.g, 100 ng, and 10 ng, respectively, of phaseolin purified from bean cotyledons as markers for quantitation; lanes 4 to 9 contain 50 ^g each of total protein extracts from leaves of transgenic tobacco plants. Plants were transformed with plasmid without inserts (Co), wild-type phaseolin (wt), T343F (two independent transformants), or A363 (two independent transformants). The arrowhead indicates the position of A363; the vertical bar indicates phaseolin fragmentation products. cause further treatment of the insoluble material with SDScontaining buffer at 90°C did not result in the recovery of additional amounts of the recombinant protein (data not shown). A363 constitutes ~0.05 to 0.1 % of total leaf protein in the transgenic plants, as indicated by quantitative comparisons with known amounts of phaseolin extracted from bean cotyledons and loaded on the same gel (Figure 2, lanes 1 to 3; the banding pattern of natural phaseolin is complex because the protein is produced by a multigene family and has different extents of glycosylation; see also below). For controls, we also produced transgenic tobacco expressing two assembly-competent forms of phaseolin: the wild-type (3-31 phaseolin (Slightom et al., 1985), from which A363 was derived, and a mutant of (3-31 phaseolin, T343F, in which Thr-343 was mutated to Phe, and thus, the consensus for N-glycosylation of Asn-341 was destroyed (Figure 1). Although it is a mutated protein, T343F can be considered to be very similar to a natural form of phaseolin, because normally in bean cotyledons, glycosylation at Asn-341 is inefficient and a significant proportion of polypeptides has therefore only a single glycan on Asn-252 (Sturm et al., 1987). Singly glycosylated phaseolin is fully competent for trimerization and delivery to protein storage vacuoles. Polypeptides of the expected molecular masses for fully glycosylated wild-type phaseolin and singly glycosylated T343F (51 and 46 kD, respectively), together with fragments in the 20- to 25-kD range, were detected in the transgenic plants (Figure 2, lanes 4 to 6). Fragmentation into polypeptides corresponding roughly to subunit halves is a common event when phaseolin is expressed in transgenic plants, al- Quality Control in the Secretory Pathway As shown in Figure 3A, A363 was located exclusively in sucrose gradient fractions that also contained BiP in organelles with a density of ~1.17 g/mL. The top fractions of the gradient contain soluble cytosolic and vacuolar proteins, because vacuoles break during homogenation; consistently, the fragments of assembly-competent phaseolin, which are vacuolar because fragmentation occurs in the vacuole (Pueyo et al., 1995), are recovered entirely in the top fractions of these gradients (data not shown). A very small proportion of A363 was recovered on top of the gradients, but this was less than the proportion of BiP, an ER resident, in the same fractions. We cannot exclude that this proportion of A363 is vacuolar; however, the presence of BiP suggests that it might result from the release of part of the soluble lumenal content of the ER or other compartments during homogenation. When homogenation and centrifugation were performed in the presence of EDTA rather than magnesium to release ribosomes from the ER and therefore lower its density but not that of the other organelles, both BiP and A363 phaseolin shifted to fractions of lighter density (Figure 3B). The results presented in Figure 3 indicate that the bulk of A363 is not located in the vacuole and is possibly located in though it does not occur in bean seeds (Bagga et al., 1992; Pueyo et al., 1995). The results shown in Figure 2 indicate that A363 does not give rise to immunoreactive fragmentation products and that its accumulation is higher with respect to unfragmented assembly-competent phaseolin. The overall amount of phaseolin, including the fragments, does not seem very different between assembly-competent and defective constructs. To obtain information on the subcellular location of A363, leaves from transgenic plants were homogenized in the presence of sucrose and absence of detergent, and organelles were separated on isopycnic sucrose gradients. Gradient fractions were then analyzed using a mixture of anti-phaseolin and anti-BiP antisera. The ER-resident chaperone BiP was used as a marker for the ER. The anti-BiP antiserum was produced against a recombinant fusion protein containing a C-terminal portion of tobacco BiP. This anti-BiP antiserum recognizes a band of the expected size (78 kD) on protein blots or by immunoprecipitation of in vivo radioactively labeled proteins (see below) and does not immunoprecipitate other members of the heat shock protein 70 family (data not shown). 1.11 1871 1.17 1.24 total Mg:2+ B EDTA Figure 3. Subcellular Distribution of Assembly-Defective Phaseolin. Leaves from transgenic tobacco expressing A363 phaseolin were homogenized, and the homogenate was fractionated on isopycnic sucrose gradient. (A) Magnesium-containing buffer. (B) EDTA-containing buffer. Gradient fractions were analyzed by SDS-PAGE followed by protein gel blotting and immunodetection with a mixture of anti-phaseolin and antiBiP antisera. In (A) and (B), the lane at right contains total unfractionated homogenate. The positions of BiP (asterisks) and A363 phaseolin (arrowheads) are indicated. Numbers at top indicate density (grams per milliliter). 1872 The Plant Cell the ER. However, it is possible that other organelles aggregate with the ER during homogenation and therefore also shift their position in the presence of EDTA. To test this possibility, we performed immunodetection analysis of isopycnic gradients using an antiserum that recognizes complex N-linked glycans of plant glycoproteins (Laine et al., 1991). Because N-linked oligosaccharide chains acquire a complex structure in the Golgi complex, immunoreactive polypeptides should not shift their position in the presence of EDTA if the Golgi complex and post-Golgi complex compartments 1.24 T and vesicles of the secretory pathway do not aggregate with the ER. The antiserum recognizes many polypeptides in plant cells (Laine et al., 1991). Part of the immunoreactive polypeptides remains on top of the gradients and most probably represents proteins that have reached the vacuole or the cell wall, whereas others are associated with membrane-bound compartments (Figure 4). The position of these along gradients containing magnesium or EDTA was very similar (compare Figures 4A and 4B). We conclude that the subcellular location of A363 corresponds to the ER or a closely related compartment. B EDTA A363 Phaseolin Is Degraded Independently of Golgi Complex-Mediated Vesicular Traffic The location of A363 indicates that the defective phaseolin is either not competent for transport beyond the ER or very rapidly degraded when it arrives at the vacuole or some other post-ER compartment. Therefore, we investigated its stability by pulse-chase labeling experiments under normal conditions or under conditions that inhibit vesicular trafficking. Protoplasts were prepared from leaves of transgenic plants expressing A363 or T343F and were subjected to 2 hr of pulse labeling in the presence of 35S-labeled amino acids and to 4 or 9 hr of chase. Protoplasts were homogenized at the end of the pulse or the chase time points. Phaseolin was immunoprecipitated and analyzed by SDS-PAGE and fluorography. The results are shown in Figure 5. The polypeptides with molecular masses of ^60 and 40 kD, present in all of the lanes of the gel, were also selected from pDHA-transformed plants (data not shown) and therefore represent endogenous immunoreactive proteins. Newly synthesized A363 consists of a major polypeptide with an M, of 46 kD and a minor polypeptide with an M, of 43 kD (Figure 5, lane 7). When synthesis was allowed to occur in the presence of the N-glycosylation inhibitor tunicamycin, A363 was synthesized as a single polypeptide with an M, of 40 kD, confirming that the two polypeptides synthesized in normal conditions represent fully and singly glycosylated A363 (data not shown). During the chase, there was a decrease in the recovery of A363 (Figure 5, lanes 7 to 9). This was not due to secretion, because A363 was not detectable in the protoplast incubation medium at any pulse-chase time point (data not shown). No fragmentation products could be detected, indicating that either full degradation or fragmenta- Figure 4. Subcellular Distribution of Glycoproteins with Complex Glycans. Leaves from transgenic tobacco plants expressing A363 phaseolin were homogenized, and the homogenate was fractionated on isopycnic sucrose gradient. (A) Magnesium-containing buffer. (B) EDTA-containing buffer. Gradient fractions were analyzed by SDS-PAGE followed by protein gel blotting and immunodetection with the anti-complex glycan antiserum. Numbers at top indicate density (grams per milliliter). tion into nonimmunoreactive peptides occurs without the formation of long-lived intermediates. This is probably related to the high susceptibility of monomeric phaseolin to degradation once attacked by proteases, as shown by in vitro experiments (Ceriotti et al., 1991). Because fragmentation of assembly-competent phaseolin in heterologous cells occurs in the vacuole, it can indicate a lower limit for the rate of phaseolin transport to this compartment. Assembly-competent T343F has an Mr of ~46 kD, and as expected, upon the chase, it was fragmented into polypeptides with molecular masses of 20 to 25 kD (Figure 5, lanes 1 to 3). The rate of fragmentation of T343F was much higher than was the rate of degradation of A363 (Figure 5, compare the disappearance of intact phaseolin in lanes 1 to 3 and 7 to 9). Figure 6 shows a detailed analysis of the kinetics of degradation of the two constructs, performed by labeling protoplasts for 1 hr followed by 2, 4, 8, or 16 hr of chase. At the normal temperature of 25°C, fragmen- Quality Control in the Secretory Pathway tation of T343F was almost twice as fast as degradation of A363. These results indicate that despite the higher susceptibility of monomeric phaseolin to in vitro proteolytic attack (Ceriotti et al., 1991), in vivo degradation of A363 is a slow process, slower than fragmentation of trimeric phaseolin, which requires transport to the vacuole. This suggests that A363 is not competent for rapid transport to the vacuole. To determine whether vesicular transport along the secretory pathway is necessary for the degradation of assemblydefective phaseolin, we performed pulse-chase labeling under conditions that inhibit activity of the secretory pathway. Brefeldin A is a fungal antibiotic that inhibits Golgi-mediated vesicular traffic. In plant cells, brefeldin A blocks both protein secretion and transport of soluble proteins to the vacuole (Gomez and Chrispeels, 1993; Jones and Herman, 1993). In the presence of brefeldin A, assembly-competent and assembly-defective phaseolin had strikingly different behaviors during the chase: fragmentation of T343F was inhibited (Figure 5, compare lanes 4 to 6 and 1 to 3), as expected if a block in transport to the vacuole occurs, whereas degrada- T343F A363 BFA: h r o f chase: 0 4 9 0 4 9 0 4 9 0 4 9 976846- 30- 143 4 5 6 8 9 10 11 12 Figure 5. Synthesis and Stability of Phaseolin Constructs under Different Conditions. Protoplasts prepared from transgenic tobacco plants expressing T343F or A363 phaseolin were pulse-labeled for 2 hr with 35Smethionine and 35S-cysteine and subjected to chase for 0, 4, or 9 hr. The pulse-chase experiment was performed in the presence (+) or absence (-) of brefeldin A (BFA). Phaseolin was immunoprecipitated and analyzed by SDS-PAGE and fluorography. The positions of BiP (asterisk), fully and singly glycosylated A363 (arrowheads), and endogenous tobacco polypeptides (dots) are indicated at right. Numbers at left indicate the positions of molecular mass markers in kilodaltons. 1873 150 10 12 14 16 18 20 hours of chase Figure 6. Rates of Degradation of Phaseolin Constructs. Protoplasts prepared from transgenic tobacco plants expressing T343F or A363 phaseolin were pulse-labeled at 25°C for 1 hr with 35 S-methionine and 35S-cysteine and subjected to chase for 0, 2, 4, 8, or 16 hr at 25°C or for 4 hr at 42°C. Phaseolin was immunoprecipitated and analyzed by SDS-PAGE and fluorography. The fluorograph was subjected to densitometric analysis, and intact phaseolin at each chase point is expressed as the percentage of the amount measured at the end of the pulse. Error bars indicate the standard deviation of results from two independent experiments. tion of A363 was unaffected (Figure 5, compare lanes 10 to 12 and 7 to 9). Heat shock inhibits trafficking of the vacuolar storage protein phytohemagglutinin beyond the ER in bean cbtyledons (Chrispeels and Greenwood, 1987) and severely decreases protein secretion in gibberellin-treated barley aleurone layers (Grindstaff et al., 1996). As shown in Figure 6, when protoplasts pulse-labeled for 1 hr at normal temperature (25°C) were subjected to chase for 4 hr at 42°C, fragmentation of T343F was inhibited, whereas degradation of A363 was slightly accelerated. From the results shown in Figures 5 and 6, we conclude that both constructs undergo proteolysis. Fragmentation of assembly-competent phaseolin requires vesicular transport to the vacuole; however, degradation of assembly-defective phaseolin is independent of Golgi complex-mediated vesicular traffic and occurs at a rate that is markedly slower than is fragmentation of trimeric phaseolin. Retention of Assembly-Defective Phaseolin Occurs before the Medial frans-Golgi Complex To investigate in more detail the pathway followed by assembly-defective phaseolin, we determined its resistance to endoglycosidase H. The enzyme removes from glycoproteins the N-linked glycans of the high-mannose type but not 1874 The Plant Cell of the complex type. Because the complex structure is acquired when acted on by glycosidases and glycosyltransferases located in the medial and frans-Golgi complex, endoglycosidase H resistance indicates that a glycoprotein has reached these compartments of the secretory pathway (Vitale and Chrispeels, 1984; Fitchette-Laine et al., 1994). As mentioned above, wild-type phaseolin exists in bean cotyledons in two glycoforms. During passage of phaseolin through the Golgi complex, the glycan of the singly glycosylated form acquires a complex structure, but the glycans of the fully glycosylated form remain of the high-mannose type (Sturm et al., 1987). Interactions between the two glycans probably make them inaccessible to the processing enzymes of the Golgi complex. Glycan processing can therefore be used as a marker for intracellular transport of singly glycosylated but not fully glycosylated phaseolin. We have shown in Figure 5 that A363 is mainly glycosylated at both sites: it is very unlikely to acquire endoglycosidase H resistance even if transported through the Golgi complex. Therefore, we introduced a stop codon at position 360 in T343F to produce an assembly-defective, singly glycosylated construct (T343FA360, schematically represented in Figure 1). The construct was expressed transiently in tobacco mesophyll protoplasts, where, as expected, it remained monomeric (data not shown), and processing of its glycan was analyzed by endoglycosidase H digestion, as shown in Figure 7. The polypeptides with molecular masses larger than T343FA360 (migrating above the position indicated by the arrowhead in Figure 7) are contaminants selected also from protoplasts transiently transformed with the plasmid without insert (data not shown). T343FA360 remained fully susceptible to endoglycosidase H during the chase, when protoplasts were cultured under normal conditions (Fig- BFA: hr of chase: endo H: 1 3 4 5 6 7 8 Figure 7. Processing of the Oligosaccharide of T343FA360 Phaseolin. Tobacco leaf protoplasts transiently expressing T343FA360 phaseolin were pulse-labeled for 2 hr with 35S-methionine and 35S-cysteine and subjected to chase for 0 or 5 hr. The pulse-chase was performed in the presence (+) or absence (-) of brefeldin A (BFA). Phaseolin was immunoprecipitated and incubated in the presence of endoglycosidase H (endo H [+]) or in the absence (-) of the enzyme as a control. Proteins were analyzed by SDS-PAGE and fluorography. The positions of intact (arrowhead) or deglycosylated (arrow) T343FA360 are indicated. ure 7, lanes 1 to 4), but it acquired complete resistance to the enzyme in brefeldin A-treated protoplasts (Figure 7, lanes 5 to 8). Therefore, in plant cells, brefeldin A not only blocks transport beyond the Golgi complex but also either promotes transport from the ER to the Golgi complex or, perhaps more likely, retains and relocates enzymes of the Golgi complex into the ER. The results indicate that the conformation of T343FA360 allows access to Golgi complexprocessing enzymes, but under normal conditions, such enzymes are not encountered by T343FA360. We conclude that the mechanism retaining assembly-defective phaseolin from trafficking does not involve recycling from the medial frans-Golgi complex. Assembly-Defective Phaseolin Is a Major BiP Ligand in Transgenic Plants, and Association with the Chaperone Is on the Pathway for Degradation of the Defective Protein We have previously shown that A363 interacts with BiP during transient expression in tobacco protoplasts (Pedrazzini et al., 1994). We have used transgenic tobacco plants expressing A363 to analyze more precisely the extent of its interactions with BiP. When protoplasts were labeled and immunoprecipitation was performed using anti-phaseolin antiserum, BiP was coselected with A363. This is visible in Figure 5. BiP has a very slow turnover, and the amount of radioactive BiP coselected is constant throughout the pulse-chase experiment. This strongly suggests that radioactive BiP synthesized during the pulse remains available for association with unlabeled A363 synthesized during the chase. Assembly-competent phaseolin rapidly forms trimers in tobacco protoplasts, and trimerization conceals BiP binding sites of phaseolin (Pedrazzini et al., 1994; Vitale et al., 1995). Consistently, there is almost no BiP associated with T343F (Figure 5). BiP has ATPase activity, and the association between BiP and its ligands is ATP sensitive (Pedrazzini and Vitale, 1996). Figure 8, lanes 1 and 2, shows that BiP coimmunoprecipitated with A363 expressed in transgenic tobacco is released when the complex is treated with ATP, indicating that the coselection is entirely related to the chaperone function of BiP. Immunoselection of labeled protoplasts using antiserum raised against tobacco BiP resulted in the ATP-sensitive coselection of A363, whereas no polypeptides were selected using the preimmune serum (Figure 8, lanes 9 to 11). A363 is the most intensely labeled polypeptide coselected with BiP in an ATP-sensitive fashion after pulse-labeling. Taking into consideration that A363 has only three methionine and no cysteine residues, we conclude that the assembly-defective phaseolin is a major BiP ligand in tobacco leaf protoplasts. The 60-kD polypeptide of unknown identity, which was selected by the anti-phaseolin antiserum, was also selected by the anti-BiP antiserum (Figure 8, lanes 3 to Quality Control in the Secretory Pathway antiserum: h r o f chase: ATP: cxPHSL aBiP P aPHSL 00 0 3 6 0 3 6 - + . . . . . . 1875 aBiP 686846- 3030- 12 3 4 5 6 7 8 9 10 11 Figure 8. Association between Assembly-Defective Phaseolin and BiP. Protoplasts from transgenic tobacco plants expressing A363 phaseolin were pulse-labeled for 1 hr with 35S-methionine and 35S-cysteine and subjected to chase for 0, 3, or 6 hr. The homogenates were selected by using anti-phaseolin (uPHSL), anti-BiP (aBiP), or preimmune (P) antiserum. After immunoselection, the material in lanes 1,2, 10, and 11 was treated with buffer supplemented with magnesium and ATP (+) or magnesium alone (-) as a control. Analysis was by SDS-PAGE and fluorography. The arrowheads and the asterisks mark the positions of fully glycosylated A363 phaseolin and of BiP, respectively. Numbers at left of gels indicate the positions of molecular mass markers in kilodaltons. Lanes 1 and 2, 3 to 8, and 9 to 11 are from independent experiments. 8; see also Figure 5). However, the significance of this coselection is unclear because it varies between different experiments (Figure 8, compare lanes 3 and 10) and is insensitive to ATP (Figure 8, compare lanes 10 and 11). In the experiment whose results are shown in lanes 3 to 8 of Figure 8, equal amounts of A363-expressing protoplasts were selected with anti-BiP (lanes 3 to 5) or anti-phaseolin (lanes 6 to 8) antiserum. The results indicate that only a proportion of defective phaseolin is coselected with BiP and vice versa. Quantitation by densitometry of the fluorographs indicates that at the end of the pulse, ~10% of total newly synthesized A363 is associated with BiP and ~15% of total newly synthesized BiP is associated with A363. The results shown in lanes 3 to 5 of Figure 8 also indicate that BiP-associated A363 decreases during the chase, as total A363 is degraded. A detailed comparison between the rates of disappearance of total and BiP-bound A363 was performed, with protoplasts subjected to 1 hr of pulse followed by 0, 2, 4, or 8 hr of chase. As shown in Figure 9, there is an initial decrease in the ratio between BiP-associated and total A363; however, subsequently, the time course of degradation of total A363 is similar to that of release of BiP-associated A363 from the chaperone. This indicates that association to BiP is on the pathway for A363 degradation and suggests that BiP-bound A363 is not a subset of molecules with a distinct destiny but rather that there is only one pool of A363 that may undergo cycles of binding and release from BiP before being degraded. 120 100 4 6 hours of chase Figure 9. Rates of Degradation of Total and BiP-Associated A363 Phaseolin. Protoplasts from transgenic tobacco plants expressing A363 phaseolin were pulse-labeled for 1 hr with 35S-methionine and 35S-cysteine and subjected to chase for 0, 2, 4, or 8 hr. Phaseolin or BiP was immunoprecipitated from equal numbers of protoplasts and analyzed by SDS-PAGE and fluorography. Total phaseolin immunoprecipitated with the anti-phaseolin antiserum or phaseolin coselected with the anti-BiP antiserum was quantified by densitometry of the fluorograph, and phaseolin at each chase point is expressed as a percentage of the amount measured at the end of the pulse. 1876 The Plant Cell DlSCUSSlON The data presented here indicate that a defect in the assembly of phaseolin results in an alteration of its intracellular transport. The following evidence indicates that intracellular traffic of assembly-defective phaseolin along the secretory pathway is blocked at some stage before the media1 tmnsGolgi stacks: (1)the defective protein is located in a subcellular compartment that is the ER or is closely associated with it; (2) its degradation is not inhibited by brefeldin A or heat shock, whereas both treatments block transport of assembly-competent phaseolin to the vacuole; (3)T343FA360 remains sensitive to endoglycosidase H after a chase; (4) A363 is a major BiP ligand in transgenic tobacco and has prolonged interactions with this ER-located chaperone; and (5) despite the fact that monomeric phaseolin is much more susceptible to in vitro proteolysis than is the assembled protein (Ceriotti et al., 1991),the rate of degradation of A363 is approximately half the rate of fragmentation of trimeric phaseolin, which occurs when transported to the vacuole, and the levels of accumulation of A363 and trimeric phaseolin are similar. Quality Control in the Secretory Pathway of Plant Cells We have presented compelling evidence for a causal relationship between the acquisition of quaternary structure and trafficking along the secretory pathway to the vacuole of plant cells. Acquisition of the correct conformation is also a prerequisite for the intracellular trafficking of many secreted and plasma membrane mammalian proteins and at least two yeast-soluble vacuolar proteins (Finger et al., 1993;Hammond and Helenius, 1995).Therefore, it appears that a conserved quality control mechanism or severa1 mechanisms controlling similar processes exist in the secretory pathway of eukaryotic cells. To date, evidence for ER quality control in higher plants has been only indirect and not definitive. lndirect evidence includes the discovery that the ER of all eukaryotes is provided with a set of very abundant resident proteins, such as BiP, protein disulfide isomerase, calnexin, calreticulin, and endoplasmin, that are involved in folding and assembly of newly synthesized polypeptides (Hammond and Helenius, 1995; Denecke, 1996). It is thought that interaction with such helpers may be part of the quality control: clearly, newly synthesized polypeptides must be confined to the ER when bound to ER residents, and plant proteins undoubtedly should not be an exception. Studies on the expression of subunits of immunoglobulins and of the maize storage proteins in transgenic plants showed that accumulation is enhanced when partner subunits are expressed together, also suggesting the presence of ER quality control in plants (Hiatt et al., 1989;Coleman et al., 1996). It has also been observed that certain mutated constructs of barley aleurain, a vacuolar protein, fail to reach the vacuole or the cell surface in transformed tobacco protoplasts (Holwerda et al., 1992).Finally, an assembly-defective mutant of pea vicilin, a storage protein closely related to phaseolin, accumulates at particularly low levels and is not located in the vacuoles in transgenic tobacco (Kermode et al., 1995).The pathways for degradation of assembly-defective phaseolin and of the above-mentioned proteins could be similar. Retention Folding and assembly usually occur in the compartment of residence of a protein, and in this sense, the ER is an exception. It has the unique characteristic of taking care of these processes for proteins that are destined for other locations. By retaining proteins in the ER, quality control probably both optimizes folding and assembly and avoids expression of defective proteins in their compartment of destination. We have shown here that unassembled phaseolin is retained in an early compartment of the secretory pathway that has low hydrolytic activity rather that being transported to the vacuole, where it would most likely be degraded quickly. Trimerization of wild-type phaseolin both in bean cotyledonary cells and in transfected tobacco protoplasts is a very rapid and efficient process, much faster than vesicular transport out of the ER (Pedrazini et al., 1994;Vitale et al., 1995).For wild-type phaseolin, it is impossible to establish whether some form of ER retention of monomers occurs before assembly. The results shown here, however, suggest that such retention is likely to occur and might favor efficient assembly. Assembly-defective phaseolin is a major newly synthesized BiP ligand in the transgenic plants that we have produced. However, we have only been able to detect 40% of A363 in association with BiP. Because we also found that 6iP-bound and total A363 have very similar degradation kinetics, we conclude that BiP binding is on the pathway to A363 degradation and that degradation probably occurs after cycles of binding and release from the chaperone. If we take into consideration that other chaperones are present in the ER and probably act on phaseolin (Lupattelli et al., 1997) and that assembly-defective phaseolin is found in a monomeric state and not as a large aggregate that might physically be unable to enter vesicular trafficking, then the prolonged interaction with chaperones could be the most reasonable explanation for retention, and a mechanism sensing such prolonged interaction could be the system leading to degradation. Degradation When defects in the newly synthesized proteins occur or when stress conditions do not allow proper structural maturation to take place, quality control eventually degrades the Quality Control in the Secretoty Pathway defective proteins, allowing the cell to maintain homeostasis (Hammond and Helenius, 1995). Similarly, we have shown that the accumulation of A363 phaseolin is not indefinitely prolonged and that the defective protein is slowly degraded. In most cases, it is still unclear which features of defective proteins make them selected substrates for degradation. A cysteine residue that mediates assembly of immunoglobulin M also promotes retention and degradation of the excess of unassembled subunits that is normally synthesized in plasma cells (Fra et al., 1993). Phaseolin, however, does not contain cysteine residues. The mechanism of degradation must sense either the prolongation of the unassembled state beyond a “physiological” period of time or the structural features of the mutant we have produced that are not present in the assembly-competent protein. This degradation is not simply caused by prolonged retention in the ER or in some undefined pre-Golgi complex compartment but requires a structural defect in phaseolin, because in the presente of brefeldin A, T343F is stable. The defect must be severe to allow for degradation: T343F can be considered a form of wild-type phaseolin, because indeed many wild-type phaseolin polypeptides do not acquire a glycan on Asn-341 (Sturm et al., 1987); however, when we treated protoplasts with tunicamycin and brefeldin A at the same time, the absence of the glycan at Asn-252, which is always present in natural phaseolin, did not affect the stability of T343F (data not shown). It has been shown recently that at least in some cases, degradation by quality control involves retrotranslocation into the cytosol and the action of the cytosol-located ubiquitin-proteasome system (Lord, 1996). This suggests that defective proteins must be unfolded, possibly with the help of chaperones, to make their pathway backward into the cytosol through a channel that could be the same channel used during synthesis and translocation into the ER or another channel not yet discovered. However, at present, we have no proof that A363 is retrotranslocated into the cytosol. An alternative pathway cannot be excluded; this could involve degradation in the ER itself or in the vacuole, reached by a mechanism that does not involve the Golgi complex. The wheat storage protein gliadin forms protein bodies in the ER, and these are then in part directly internalized into storage vacuoles by autophagy (Galili et al., 1996). The tonoplast intrinsic protein TIP expressed in tobacco mesophyll protoplasts reaches its destination through a brefeldin Ainsensitive process (Gomez and Chrispeels, 1993). However, it is not yet known whether the Golgi complex-independent pathways of gliadin and TIP to the vacuole are also insensitive to heat shock, as is the case with A363 degradation. 1877 port, but the mutated phaseolin is much more unstable than is the wild-type counterpart once it reaches the storage vacuoles of transgenic tobacco seeds, suggesting that its conformation has been altered (Hoffman et al., 1988; Pueyo et al., 1995). In addition, fully unglycosylated phaseolin is considerably less stable than is glycosylated phaseolin when expressed in transgenic tobacco seeds, and again, instability is due to degradation in protein storage vacuoles (Bustos et al., 1991). Trimerization of phaseolin is unaffected by the lack of glycans (Vitale et al., 1995) and is only partially affected by the introduction of the methionine-rich peptide (Hoffman et al., 1988). Clearly, quality control fails to recognize these phaseolin mutants as defective, although they are defective in their function of stable storage material. Again, we can reason that trimer formation is a key step that conceals most if not all interactions with the retention mechanism, which may be constituted by the chaperone machinery. This would explain why defective phaseolins that can trimerize escape quality control. We have shown previously that the BiP binding sites in phaseolin are concealed upon assembly, independent of the presence of the glycans (Vitale et al., 1995). We are currently attempting to identify regions necessary for BiP binding in phaseolin monomers. If we succeed, the hypothesis that binding to chaperones mediates retention can be tested by fusing such regions to reporter secretory proteins. METHODS Phaseolin Mutants All phaseolin constructs derive from the cDNA clone p31 (Slightomet al., 1985). The constructions of A363 and T343F have been described by Ceriotti et al. (1991) and Lupattelli et al. (1997), respectively. To produce T343FA360, we introduced a stop codon at position 360 of T343F by site-directed mutagenesis using the antisense oligonucleotide 5’-ATTGTCCGTeCCTGCAAG-3’ (where underlining indicates the antisense for the stop codon). Mutagenesis was performed with a fragment subcloned in the vector M13mp18. The Sculptor in vitro mutagenesis system (Amersham) was used for mutagenesis, and the fragment containing the mutated sequence was fully sequenced before being reintroduced into phaseolin cloned in the plasmid pDHA (Pedrazzini et al., 1994; Tabe et al., 1995) for constitutive expression in plant cells. Phaseolin constructs in the pDHA vector were used for transient expression in protoplasts. For constitutive expression in transgenic tobacco, the pDHA vector without inserts or containing wild-type, T343F, or A363 phaseolin was inserted into the Hindlll site of the binary vector pGA470 (An et al., 1985),which was then used to transform strain LBA4404 of Agrobacterium tumefaciens by triparental mating. Accuracy of the Recognition Mechanism How accurate is quality control? The introduction of a peptide enriched in methionine residues in the structure of phaseolin does not make the protein incompetent for trans- Transformation of Tobacco Luria-Bertani medium (5 mL) containing 20 pg/mL rifampicin (Sigma) and 12.5 pg/mL tetracycline (Sigma) was inoculated with a single 1878 The Plant Cell colony of the different transformed agrobacteria and incubated at 29°C for 2 days. The cultures were diluted 1:100, incubated overnight of l), and diluted 150 in shootto obtain 108 cells per mL (OD, inducing medium (SIM; Murashige and Skoog salts containing 0.5 mg/L 6-benzylaminopurine; Sigma). This suspension was used to transform leaf discs from Nicotiana tabacum cv SR1 by cocultivation in 10-cm Petri dishes (20 leaf discs per dish containing 10 mL of suspension) for 48 hr at 28°C in the dark (An et al., 1986).After cocultivation, bacterial cells were washed off with SIM containing 250 mg/L cefotaxime, and the leaf discs were grown at 25°C under full light on SIM containing agar, 250 mg/L cefotaxime, and 1O0 mg/L kanamycin (10 leaf discs per 10-cm Petri dish). After 5 weeks, shoots were plated on half-strengthMurashige and Skoog salts, 100 mg/L kanamycin, and 250 mg/L cefotaxime until the new plants developed. Transformed plants were grown at 25°C in 16 hr of light in axenic culture in Magenta GA-7 vessels (Sigma)without antibiotics and propagated every 5 to 6 weeks. Production of the Anti-BiP Antiserum The sequence encoding amino acids 551 to 667 of tobacco BiP clone NTBLP4 (Denecke et al., 1991) was amplified by polymerase chain reaction. The amplified sequence was fused in frame to the 3’ end of the mal€ gene in the expression vector pMAL-c2 (Riggs, 1992), generating plasmid pMALBiP. The fscherichia c06 XL1-Blue transformed with pMALBiP plasmid was cultured in 400 mL of LuriaBertani medium, and expression of the fusion protein was induced by isopropylp-D-thiogalactopyranoside treatment for 2 hr. Total bacteria1 proteins were extracted by sonication and fractionated on an amylose resin column (1.5 X 10 cm), as described by Riggs (1992). The fusion protein was retarded but not retained by the column; fractions eluting from the amylosecolumn and containing the fusion protein were then pooled, dialyzed against 100 mM NaCl and 20 mM Tris, pH 7.5, and loaded onto a DEAE-Sephacel (Pharmacia, Uppsala, Sweden) column (1.5 x 7 cm) at 4°C. The resin was extensively washed with the same buffer and then with 30 mL of 500 mM NaCl and 20 mM Tris, pH 7.5, collecting fractions of 2 mL. An aliquot of each fraction was analyzed by SDS-PAGE, and fractions containing the purifiedfusion protein were pooled and concentrated to 0.75 mg/ mL in PBS using Centriplus 10 (Amicon, Beverly, MA) concentrators. The concentrated protein was used to immunize rabbits. Total Leaf Protein Extraction, Subcellular Fractionation, and Protein Gel Blot Analysis For analysis of total proteins, leaves from transformed plants were homogenized in an ice-cold mortar with 0.2 M NaCI, 1 mM EDTA, 2% p-mercaptoethanol, 0.2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 0.1 M Tris-CI, pH 7.8, supplemented with a Complete (Boehringer Mannheim) protease inhibitor cocktail. The buffer-to-tissue ratio was 6:l. The homogenate was centrifuged at 15009 at 4°C for 10 min, and the supernatant was analyzed by SDS-PAGE on 15% acrylamidegels. lmmunodetectionof phaseolin and BiP in protein gel blots was performed using the enhanced chemiluminescence(ECL) system (Amersham)following the manufacturer’s protocol, using rabbit polyclonal antisera raised against phaseolin purified from bean cotyledons (D’Amico et al., 1992), against plant complex N-linked glycans (Lainé et al., 1991), or against a recombinant MAL-BiP fusion protein (see above). Subcellular fractionation on isopycnic sucrose gradients in the presence of EDTA or magnesium was performed as described by Ceriotti et al. (1995). Protoplast Preparation, Pulse-Chase Labeling, Immunoprecipitation, and Endoglycosidase H Digestion Protoplasts were prepared from leaves (5 to 7 cm long), as described by Pedrazzini et al. (1994). Radioactivelabeling was performed by incubation at 25°C in the dark in K3 medium (Gamborg’sB5 basal medium with minimal organics [Sigma], supplemented with 750 mg/L CaC1,~2H,O, 250 mg/L NH,NO, 136.2 g/L sucrose, 250 mg/L xylose, 1 mg/L 6-benzylaminopurine, and 1 mg/L a-naphthalenacetic acid, pH 5.5) supplemented with 150 pg/mL of BSA and 1O0 pCi/mL of PRO-MIX (a mixture of 35S-methionineand cysteine; Amersham). The protoplast concentration was 106cells per mL. Chase was performed by adding unlabeled methionine and cysteine to 10 mM and 5 mM, respectively, from a x10 concentrated stock. In some experiments, before radioactive labeling, protoplasts were incubated for 45 min in K3 medium supplemented with 10 pg/mL brefeldin A (Boehringer Mannheim; stock solution of 2 mg/mL in ethanol stored at -20°C) or 50 pg/mL tunicamycin (Boehringer Mannheim; stock solution of 5 mg/mL in 10 mM NaOH stored at 4°C) and maintained under the same conditions for the entire experiment. At the desired time points, 3 volumes of ice-cold W5 medium (154 pM NaCI, 5 pM KCI, 125 pM CaCI2.2H,O, 5 pM glucose) was added, and samples were centrifuged for 5 min at 509. The supernatant, containing secreted proteins, was removed, leaving 100 pL to cover the protoplasts. The supernatant and protoplasts were frozen separately in liquid nitrogen and stored at -80°C. Homogenizationwas performed by adding to the frozen samples 2 volumes of ice-cold protoplast homogenizationbuffer (150 mM TrisCI, 150 mM NaCI, 1.5 mM EDTA, and 1.5% Triton X-100, pH 7.5) supplemented, immediately before use, with 1.5 mM phenylmethylsulfonyl fluoride and the complete protease inhibitor cocktail. After brief vortexing, the samples were used for immunoprecipitation, as described by D’Amico et al. (1992), using the antisera mentioned above. In some cases, the immunoprecipitatewas treated with ATP, as described by Vitale et al. (1995). Digestion of immunoprecipitated proteins with endoglycosidase H (recombinant; Boehringer Mannheim) was performed as described by Ceriotti et al. (1991). Radioactive samples were analyzed by SDS-PAGE on 15% acrylamide gels. Rainbow i4C-methylated proteins (Amersham) were used as molecularweight markers. Gels were treated with 2,5-diphenyloxazole dissolved in DMSO, and radioactive polypeptides were revealed by autoradiography. Transient Transformation of Protoplasts For transient expression of phaseolin constructs, protoplasts prepared from leaves of untransformed plants were subjected to polyethylene glycol-mediated transfection, as described by Pedrazzini et al. (1994),with the following modifications.The concentration of protoplasts before the addition of polyethylene glycol was 106cells per mL; no carrier herring sperm DNA was added. After overnight incubation at ZYC, transformed protoplasts were then subjected to radioactive labeling, as described above. Quantification of Radioactive Proteins and lmmunoblots After fluorography (SDS-PAGE of radioactive proteins) or ECL immunodetection of protein blots, quantificationof the relative intensities of the bands on the films was performed by microdensitometry, using a TLC Scanner II (Camag, Muttenz, Switzerland).Care was taken to use film exposures that were in the linear range of film darkening. Quality Control in the Secretory Pathway ACKNOWLEDGMENTS We thank Jürgen Denecke for providing clone NTBLP4 and Lo’ic Faye for providing the antiserum against the plant complex N-linked glycans. This work was supported in part by the European Union (Grant No. CHRX-CT94-0590) and by the Progetto Speciale Biologia e produzioni agrarie per un’agricoltura sostenibile of the Consiglio Nazionale delle Ricerche. ReceivedApril 30, 1997; accepted August 8, 1997. REFERENCES An, G., Watson, B.D., Stachel, S., Gordon, M.P., and Nester, E.W. (1985). New cloning vehicles for transformation of higher plants. EM60 J. 4,277-284. An, G., Watson, B.D., and Chiang, C.C. (1986). Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system. Plant Physiol. 81, 301-305. Bagga, S., Sutton, D., Kemp, J.D., and Sengupta-Gopalan, C. (1992). 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E Pedrazzini, G Giovinazzo, A Bielli, M de Virgilio, L Frigerio, M Pesca, F Faoro, R Bollini, A Ceriotti and A Vitale Plant Cell 1997;9;1869-1880 DOI 10.1105/tpc.9.10.1869 This information is current as of June 16, 2017 Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw15322 98X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY