Volume 8, Issue 2, Pages 228-241 (February 2015) Hsp90 Is Involved in the Regulation of Cytosolic Precursor Protein Abundance in Tomato  Bodo Tillmann, Sascha Röth, Daniela Bublak, Manuel Sommer, Ernst H.K. Stelzer, Klaus-Dieter Scharf, Enrico Schleiff  Molecular Plant  Volume 8, Issue 2, Pages 228-241 (February 2015) DOI: 10.1016/j.molp.2014.10.005 Copyright © 2015 The Author Terms and Conditions

Figure 1 Chaperone Influence on Chloroplast Protein Abundance at Steady-State Conditions. (A) Protoplasts were co-transformed with GFP, pOE33-GFP, or pSSU-GFP; Neo (lane 1) as well as increasing amounts of Hsp90-2 and counterbalancing amounts of Neo plasmid DNA. Protoplasts were harvested after 14 h and proteins were subjected to SDS–PAGE and immunodecorated with anti-GFP (panel 1), anti-profilin (for Hsp90 detection, panel 2), anti-neomycin phosphotransferase (panel 3), or anti-actin (panel 4) antibodies. (B) Protoplasts were co-transformed with the indicated constructs; Neo (Neo) and 1 μg of Hsp90-2 (lane 2) or 10 μg of Hsp90-2 (lane 3), Hsp90-IR (90 IR), Hsp70-1 (H70), or Hsp70-IR (70 IR) encoding plasmid DNA and processed as in (A). In (A) and (B) the migration of the intermediate (gray triangle) and mature form (white triangle) of the accordant protein is shown. The star indicates a cross-reaction of the antibody. (C) Protoplasts were co-transformed with the indicated preprotein and chaperone constructs. GFP fluorescence after 14 h of expression is shown for a representative image (Supplemental Figure 2). Molecular Plant 2015 8, 228-241DOI: (10.1016/j.molp.2014.10.005) Copyright © 2015 The Author Terms and Conditions

Figure 2 Time-Dependent Influence of Hsp90 on GFP and pOE33-GFP Abundance. Protoplasts were transformed with GFP (circles) and pOE33-GFP (squares) in the presence of Neo (open symbols) or Hsp90-2 expression constructs (closed symbols). The fluorescence was monitored as described in the Methods section, and the averages and standard deviations of eight independent results are shown. Molecular Plant 2015 8, 228-241DOI: (10.1016/j.molp.2014.10.005) Copyright © 2015 The Author Terms and Conditions

Figure 3 Hsp90 and Hsp70 Influence the Protein Abundance during Expression. (A) Protoplasts were co-transformed with GFP, pSSU-GFP, or pOE33-GFP and Neo (Neo), and 1 μg of Hsp90-2 (lane 2) or 10 μg of Hsp90-2 (lane 3), Hsp90-IR (90 IR), Hsp70-1 (H70), or Hsp70-IR (70 IR) plasmid DNA. Protoplasts were harvested after 7 h, and proteins subjected to SDS–PAGE and immunodecorated with anti-GFP (panel 1), anti-profilin (panel 2), anti-HA (panel 3), anti-Neo (panel 4), or anti-actin (panel 5) antibodies. (B) Protoplasts were co-transformed with pOE33-HIS construct (lanes 1–3) and 10 μg of Hsp90-2 (H90) or Hsp90-IR (90 IR) plasmid DNA, and analyzed as in (A). (C) Protoplasts were co-transformed with the indicated preprotein, Hsp expression, or IR constructs (as in (A)). Total RNA was isolated and analyzed by qRT-PCR using oligonucleotides to amplify GFP or actin transcripts. The values are normalized to the Neo sample. The standard deviation of at least three different biological replicas is shown. (D) Protoplasts were transformed with the indicated constructs. The GFP (top) and chlorophyll autofluorescence (middle) was recorded with CLSM, and the overlay of both signals is shown (bottom). Depicted are representative images. (E) Protoplasts were co-transformed with the indicated constructs and Neo (Neo), 10 μg of Hsp90-2 (lane 2), Hsp90-IR (lane 3), Hsp70-1 (lane 4), or Hsp70-IR (lane 5) plasmid DNA, respectively. Protoplasts were treated as in (A). In (A) and (C) the migration of the precursor (black triangle), intermediate (gray triangle), and mature form (white triangle) of the according protein is shown. (F) Protoplasts were co-transformed with indicated constructs and 10 μg of Hsp90-2. After expression, protoplasts were lysed and complexes immunoprecipitated with GFP antibodies. Shown is the input (IP, 25%), flow through (FT, 25%), the last wash fraction (W, 100%), and the eluate (E, 100%). The protein abundance was quantified; the values normalized to the fraction size and input are shown. Molecular Plant 2015 8, 228-241DOI: (10.1016/j.molp.2014.10.005) Copyright © 2015 The Author Terms and Conditions

Figure 4 Concentration-Dependent Action of the Chaperones. Protoplasts were co-transformed with constructs encoding the indicated proteins and Neo (neo), Hsp90-2 encoding plasmid DNA with (H90) or without TEV-enhancer (ΔT-H90), Hsp70-1 encoding plasmid DNA with (T-H70) or without TEV-enhancer (H70), or pSSU encoding plasmid DNA with (T-pSSU-Venus) or without TEV-enhancer (pSSU-GFP). Protoplasts were processed as in Figure 3A. The migration of the intermediate (gray triangle) and mature form (white triangle) of the accordant protein is shown. Molecular Plant 2015 8, 228-241DOI: (10.1016/j.molp.2014.10.005) Copyright © 2015 The Author Terms and Conditions

Figure 5 Hsp90 Influences the Abundance of Different Preproteins. (A) Protoplasts were co-transformed with mSSU-GFP, mOE33-GFP, tpSSU-mOE33-GFP, or tpOE33-mSSU-GFP and Neo (lane 1), Hsp90-2 (lane 2), or Hsp90-IR (lane 3), and treated as in Figure 3A. (B) Protoplasts were transformed with the indicated constructs. The GFP fluorescence is shown. Depicted are representative images; controls are shown in Supplemental Figure 6. (C) Protoplasts were co-transformed with plasmids coding for the precursor or mature form of the indicated proteins and Neo (lane 1), Hsp90-2 (lane 2), or Hsp90-IR (lane 3), and processed as in (A). Controls are shown in Supplemental Figures 7 and 8. Molecular Plant 2015 8, 228-241DOI: (10.1016/j.molp.2014.10.005) Copyright © 2015 The Author Terms and Conditions

Figure 6 The Influence of Hsp90 and Hsp70 on the Abundance of Different Proteins. Protoplasts were co-transformed with the indicated protein-encoding plasmids and Neo (lane 1), Hsp90-2 (lane 2), Hsp90-IR (lane 3), Hsp70-1 (lane 4), or Hsp70-IR encoding DNA (lane 5), and treated as in Figure 3A. Controls are shown in Supplemental Figure 9. Molecular Plant 2015 8, 228-241DOI: (10.1016/j.molp.2014.10.005) Copyright © 2015 The Author Terms and Conditions

Figure 7 The Solubility of Mature Preproteins. (A) Protoplasts were co-transformed with indicated constructs and Neo. Protoplasts were lysed after 7 h of expression (input, I), and insoluble (P) and soluble (S) proteins separated. Equal amounts were subjected to SDS–PAGE and Western blotting with GFP antibodies. For control, Ponceau staining is shown. (B) Protoplasts were co-transformed with indicated constructs. After expression, protoplasts were lysed and complexes immunoprecipitated with GFP antibodies. Shown is the input (IP, 25%), flow through (FT, 25%), the first and the last wash fraction (W1, W2, 100%) and the eluate (E, 100%). The complex composition was probed by antibodies against Hsp90, Hsp70, and GFP. In contrast to Figure 3F, protoplasts were not co-transformed with Hsp90-2 expression construct and, thus, only the endogenous Hsps were detected. Molecular Plant 2015 8, 228-241DOI: (10.1016/j.molp.2014.10.005) Copyright © 2015 The Author Terms and Conditions

Figure 8 The Influence of Chip and HOP on Cytosolic Protein Abundance. (A) Protoplasts were co-transformed with constructs encoding GFP, pSSU-GFP, or pOE33-GFP and Neo, HOP, Chip, or the IR construct against Hop (HOP-IR) or Chip (ChipIR). Protoplasts were processed as in Figure 3A using anti-myc antibodies to detect HOP and Chip (panels 2 and 3). The localization of Chip is demonstrated in Supplemental Figure 12. (B) Protoplasts were co-transformed with constructs encoding pSSU-GFP (left) or pOE33-GFP (right) in combination with the indicated constructs, and protoplasts were processed as in Figure 3A. The intensity of mature, intermediate, and precursor form was quantified together and the ratio calculated for each blot (see Methods section). The log2 value of the quantified band intensities and its standard deviation are shown. Experiments shown in Figures 3–7 are included in the quantification. (C) Protoplasts were co-transformed with pSSU-GFP (left) or pOE33-GFP (right) and Neo (neo), the Hsp90-2 expression construct (H90), or the Hsp90-2 and the Chip expression constructs (H90/C) in the presence of the proteasomal inhibitor MG132. Protoplasts were processed as in (A). Ponceau staining is shown as a loading control. (D) Protoplasts were co-transformed with pOE33-GFP in the absence (left) or presence (right) of the proteasome inhibitor MG132. After expression, protoplasts were solubilized and incubated with antibodies against GFP. Shown is the input (I, 10%), flow through (FT, 10%), the first and the last wash steps (W1, W5, 10%), and the elution (E, 100%), which were subjected to SDS–PAGE followed by Western blotting with antibodies against ubiquitin (top) and GFP (bottom). Molecular Plant 2015 8, 228-241DOI: (10.1016/j.molp.2014.10.005) Copyright © 2015 The Author Terms and Conditions

Figure 9 Model of the Cytosolic Regulation of Precursor Abundance. Preproteins are targeted to chloroplasts in a chaperone-dependent manner (1, 4). If import is blocked or the precursor pool exceeds the import capacity, chaperones redirect the preproteins (2, 5) toward degradation (3, 6), by CPR processes. E3 L., E3 ligase. Molecular Plant 2015 8, 228-241DOI: (10.1016/j.molp.2014.10.005) Copyright © 2015 The Author Terms and Conditions