Volume 1, Issue 6, Pages 1067-1076 (November 2008) Protein Domains Involved in Assembly in the Endoplasmic Reticulum Promote Vacuolar Delivery when Fused to Secretory GFP, Indicating a Protein Quality Control Pathway for Degradation in the Plant Vacuole  Foresti Ombretta , De Marchis Francesca , de Virgilio Maddalena , Klein Eva M. , Arcioni Sergio , Bellucci Michele , Vitale Alessandro   Molecular Plant  Volume 1, Issue 6, Pages 1067-1076 (November 2008) DOI: 10.1093/mp/ssn066 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 sGFP418 Undergoes Brefeldin A-Sensitive Post-Translational Proteolytic Processing. Protoplasts prepared from leaves of transgenic tobacco expressing sGFP418 were subjected to pulse-labeling with 35SMet and 35SCys for 30 min followed by chase for the indicated h, in the presence (+) or absence (–) of brefeldin A (BFA). At the end of each chase point, protoplasts or their incubation media were homogenated and proteins immunoprecipitated with anti-GFP antiserum. Analysis was by SDS–PAGE and fluorography. The positions of intact (arrowhead) and processed (arrow) sGFP418 are indicated on the left. Numbers on the right indicate the positions of molecular mass markers, in kDa. Molecular Plant 2008 1, 1067-1076DOI: (10.1093/mp/ssn066) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 The Processed Form of sGFP418 Does Not Interact with BiP. Protoplasts prepared from leaves of transgenic tobacco expressing sGFP418 were subjected to pulse-labeling with 35SMet and 35SCys for 30 min (A) or 1 h (B) followed by chase for the indicated h. At the end of each chase point, protoplasts were homogenated and proteins immunoprecipitated with anti-BiP or anti-GFP antiserum, as indicated. Analysis was by SDS–PAGE and fluorography. The positions of BiP (asterisk), intact sGFP418 (arrowhead), and processed sGFP418 (arrow) are indicated on the left. Numbers on the right indicate the positions of molecular mass markers, in kDa. Molecular Plant 2008 1, 1067-1076DOI: (10.1093/mp/ssn066) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Protein-Blot Analysis of sGFP418. (A) Proteins were extracted from young leaves of transgenic tobacco plants expressing sGFP-AFVY, sGFP418, or sGFPHDEL, or from untransformed plants, as indicated above each lane. Equal amounts of leaf homogenate were analyzed by SDS–PAGE followed by protein blot using anti-GFP antibodies. The positions of intact (arrowhead) and processed (arrow) sGFP418 are indicated on the left. Numbers on the right indicate the positions of molecular mass markers, in kDa. (B) Shorter exposure of the protein blot shown in (A). Molecular Plant 2008 1, 1067-1076DOI: (10.1093/mp/ssn066) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 The Processed Form of sGFP418 Is In the Vacuole. (A, B) Leaves from transgenic tobacco plants expressing sGFP418 were homogenized in buffer devoid of detergent and containing 12% (w/w) sucrose. The homogenate was subjected to centrifugation on a 16–55% (w/w) isopycnic sucrose gradient. Gradient fractions were collected and analyzed by SDS–PAGE followed by protein blot with anti-GFP (A) or anti-BiP (B) antiserum. Top of the gradients is on the left. The last lane on the right (U) contains an aliquot of unfractionated homogenate. Numbers at the top indicate density (grams per milliliter). The positions of intact (arrowhead) and processed (arrow) sGFP418 are indicated on the left. Numbers on the right indicate the positions of molecular mass markers, in kDa. (C) Leaves from transgenic tobacco plants expressing sGFPHDEL were homogenated and fractionated as in (A). Protein blot was performed with anti-GFP antiserum. (D, E) Protoplast were prepared from transgenic tobacco plants expressing sGFP418. Protoplast homogenate (lane 1) or purified vacuoles with the same (lane 2) or four-fold (lane 3) α-mannosidase activity as in the homogenate in lane 1 were analyzed by SDS–PAGE and protein blot with anti-BiP (D) or anti-GFP (E) antiserum. Numbers on the right indicate the positions of molecular mass markers, in kDa. Molecular Plant 2008 1, 1067-1076DOI: (10.1093/mp/ssn066) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Protein Blot Analysis of Zein–GFP. Proteins were extracted from leaves of transgenic tobacco plants expressing the constructs indicated above each lane or from wild-type (wt) tobacco. Extraction was performed from equal amounts of leaf tissues with buffer supplemented (R) or not (N) with 2-mercaptoethanol. The material that remained insoluble after homogenization with 2-mercaptoethanol was also analyzed (I). Proteins were analyzed by SDS–PAGE followed by protein blot using anti-γ-zein antibodies. The positions of intact zein–GFP (empty arrowhead), zeolin–Nef (filled circle) and zein–Nef (empty circle) are indicated. Numbers on the right indicate the positions of molecular mass markers, in kDa. Molecular Plant 2008 1, 1067-1076DOI: (10.1093/mp/ssn066) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Zein–GFP Undergoes Brefeldin A-Sensitive Post-Translational Proteolytic Processing. (A) Protoplasts prepared from leaves of transgenic tobacco expressing zein–GFP were subjected to pulse-labeling with 35SMet and 35SCys for 1 h followed by chase for the indicated time. At the end of each chase point, protoplasts were homogenated and proteins immunoprecipitated with anti-GFP antiserum. Homogenization and immunoprecipitation were performed in the presence (+) or absence (–) of 2-mercaptoethanol (2-ME). Analysis was by SDS–PAGE and fluorography. The positions of intact (empty arrowhead) and processed (arrow) zein–GFP are indicated on the left. Numbers on the right indicate the positions of molecular mass markers, in kDa. (B) Protoplasts prepared from leaves of transgenic tobacco expressing zein–GFP were subjected to pulse-chase as in (A) in the presence (+) or absence (–) of brefeldin A (BFA). Homogenization was performed in the presence of 2-mercaptoethanol. Immunoprecipitation was with anti-GFP antiserum. Analysis and symbols are as in (A). (C) Intensities of the fluorography bands shown in (B) were measured and expressed as a percentage of the intensity at 0 h chase, for BFA-treated or untreated samples. Molecular Plant 2008 1, 1067-1076DOI: (10.1093/mp/ssn066) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions