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First published online 29 March 2005
doi: 10.1242/jcs.02277

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Homologous and heterologous reconstitution of Golgi to chloroplast transport and protein import into the complex chloroplasts of Euglena

Silvia Sláviková^1,*,, Rostislav Vacula^1,2,*,, Zhiwei Fang^1,, Tomoko Ehara³, Tetsuaki Osafune⁴ and Steven D. Schwartzbach^1,2,¶

¹ School of Biological Sciences, E207 Beadle Center, University of Nebraska, Lincoln, NE 68588, USA
² Department of Biology, 201 Life Sciences, University of Memphis, 3700 Walker Ave, Memphis, TN 38152, USA
³ Department of Microbiology, Tokyo Medical University, 6-1-1 Shinjuku, Tokyo 160-8402, Japan
⁴ Department of Life Science, Nippon Sport Science University, 1221-1 Kamosida, Yokohama 227-0033, Japan

View larger version (41K): [in a new window]   Fig. 1. Incubation at 15°C reversibly inhibits transport of pLHCPII from the Golgi apparatus to the chloroplast. Cell-free extracts were prepared from dark-grown Euglena exposed to light at 26°C for 24 hours, incubated at 15°C or 26°C for 2 hours and pulse-labeled at 15°C or 26°C for 10 minutes with [35S]sulfate. Cells pulse-labeled at 15°C were chased for 20 or 30 minutes at 15°C or 26°C with unlabeled sulfate. Organelles were separated by isopycnic sucrose gradient centrifugation, and each gradient fraction was immunoprecipitated with antibody against Euglena LHCPII. Immunoprecipitates were analyzed on SDS gels, and gels were scanned with a PhosphorImager. To allow direct comparisons between gradients loaded with differing amounts of 35S-labeled protein, the amount of pLHCPII and LHCPII in each fraction is plotted as a percentage of the total immunoprecipitate (pLHCPII and LHCPII) recovered from the gradient. pLHCPII accumulated in the Golgi apparatus in cells maintained at 15°C and was transferred to the chloroplast and converted to mature LHCPII between 20-30 minutes after transfer to 26°C.

View larger version (67K): [in a new window]   Fig. 2 . Time-dependent in vitro reconstitution of Golgi to chloroplast transport. An isolated 35S-labeled Golgi membrane fraction (M) was incubated in the presence (+) or absence (–) of Euglena chloroplasts (Chlor) in the light for 30 or 60 minutes. Fluorographs of [35S]pLHCPII immunoprecipitated from Percoll-purified chloroplasts show pLHCPII present in the Golgi membrane fraction was transported to and imported into isolated chloroplasts where it was processed to LHCPII and a 50 kDa partially processed pLHCPII polyprotein in a time and chloroplast-dependent manner.

View larger version (71K): [in a new window]   Fig. 3. Golgi to chloroplast transport and protein import requires light and ATP. Isolated Euglena chloroplasts were incubated with a 35S-labelled Golgi membrane fraction (M) with the indicated additions (+) and omissions (–) for 60 minutes. Fluorographs of [35S]pLHCPII immunoprecipitated from Percoll-purified chloroplasts are shown. The amount of LHCPII and the 50 kDa partially processed pLHCPII polyprotein transported to and imported into isolated chloroplasts was reduced when ATP was omitted from the reaction or when the reaction was incubated in the dark indicating a requirement for an extra- and intrachloroplast source of ATP.

View larger version (35K): [in a new window]   Fig. 4. Import of proteins into Euglena chloroplasts requires intact chloroplasts and GTP hydrolysis but does not require an N-ethylmaleimide (NEM)-sensitive factor. An isolated 35S-labelled Golgi membrane fraction (M) was incubated in the light in the presence (+) or absence (–) of intact or osmotically lysed Euglena chloroplasts with (+) or without (–) addition of GTPS and with (+) or without (–) pre-treatment of the chloroplast and cytoplasmic fractions with NEM. Fluorographs of [35S]pLHCPII immunoprecipitated from Percoll-purified chloroplasts show that conversion of pLHCPII to LHCPII and the 50 kDa partially processed pLHCPII polyprotein required intact chloroplasts. pLHCPII, LHCPII and the 50 kDa partially processed pLHCPII polyprotein were not recovered with chloroplasts in reactions containing GTPS. LHCPII and the 50 kDa partially processed pLHCPII polyprotein were recovered with NEM treated reactions indicating Golgi to chloroplast transport and import required GTP hydrolysis but did not require an NEM-sensitive factor.

View larger version (60K): [in a new window]   Fig. 5. The Euglena pSSU pre-sequence region between the signal peptidase cleavage site and stop-transfer membrane anchor sequence functions as a transit peptide with pea chloroplasts. Schematic diagram (left) of pSSU deletion constructs indicating the pre-sequence (PRESEQ), hydrophobic domains (dark boxes), predicted signal peptidase cleavage site (arrow) and higher plant stromal processing peptidase processing site (arrow) respectively. In vitro synthesized 35S-labeled proteins (tran) corresponding to a one-unit pSSU encoded by plasmid 1pSSU (A) and pSSUs with pre-sequence deletions encoded by plasmids 1PSSU1-111 (B), 1pSSU289-342 (C), 1pSSU289-402 (D) and mSSU (E) were incubated with Euglena chloroplasts with the indicated additions (+) and omissions (–). Fluorographs (right) of 35S-labeled protein from Percoll-purified chloroplasts incubated with or without thermolysin show protease-protected proteins absent from the translation products (arrows) and present in the translation products (arrowheads) were recovered with chloroplasts incubated with the pSSUs lacking the pre-sequence signal peptide domain (panel B), lacking the signal peptide domain and the hydrophobic stop-transfer membrane anchor sequence (panel C) and lacking the signal peptide domain and the sequence C-terminal to the start of the hydrophobic stop-transfer membrane anchor sequence (panel D) localizing the functional transit peptide to the region between the signal peptidase cleavage site and the stop-transfer membrane anchor sequence.

View larger version (45K): [in a new window]   Fig. 6. Euglena preproteins are not post-translationally imported into Euglena chloroplasts. Schematic diagram (left) of pSSU deletion constructs indicating the pre-sequence, (PRESEQ) hydrophobic domains (dark boxes), predicted signal peptidase cleavage site (arrow) and higher plant stromal processing peptidase processing site (arrow) respectively. In vitro synthesized [35S]-labeled proteins (tran) corresponding to a one-unit pSSU encoded by plasmid 1pSSU (A) and pSSUs with pre-sequence deletions encoded by plasmids 1PSSU1-111 (B), 1pSSU289-342 (C), 1pSSU289-402 (D) and mSSU (E) were incubated with Euglena chloroplasts with the indicated additions (+) and omissions (–). Fluorographs (right) of 35S-labeled protein from Percoll-purified chloroplasts incubated with or without thermolysin show that pSSU and pSSUs with pre-sequence deletions but not mature SSU can associate with Euglena chloroplasts but can not be imported post-translationally.

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