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doi: 10.1242/10.1242/jcs.00092

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In vivo characterization of diatom multipartite plastid targeting signals

¹ Martek Biosciences Corp, 6480 Dobbin Rd., Columbia, MD 21045, USA
² Fachbereich Biologie, Universität Konstanz, Postfach M611, 78457 Konstanz, Germany
³ School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia
⁴ Carnegie Institution of Washington, Department of Plant Biology, 260 Panama Street, Stanford, CA 94305, USA.

View larger version (27K): [in a new window]   Fig. 1. Pre-sequences of fusion proteins of BiP (A) and AtpC (B) from Phaeodactylum tricornutum fused to GFP, and various fusions in which part of the AtpC pre-sequence was deleted. The signal sequences and the N-terminus of GFP are in small letters, while the transit peptide domains are shown in capital letters. The bars below represent the signal sequences (black), transit peptides (grey), N-termini of BiP and AtpC (white) and GFP (stripes). Arrows mark probable cleavage sites for signal sequences. Cleavage sites were determined by N-terminal sequencing, as described in the Results.

View larger version (44K): [in a new window]   Fig. 2. P. tricornutum expressing GFP. Light microscopical images of cells are shown on the left while their corresponding fluorescence images (GFP) are presented on the right (excitation with UV light). (A) Cytosolic expression of GFP without a targeting sequence. (B) GFP targeted to the ER (BiPGFP construct). (C) GFP targeted to the plastids (AtpC1GFP construct). Bars, 2 µm.

View larger version (26K): [in a new window]   Fig. 3. GFP accumulation in the ER of P. tricornutum expressing a BiP:GFP fusion protein. (A) Images from a confocal laser scanning microscope. Top-left: green GFP fluorescence; Top-right: red chlorophyll fluorescence indicating the location of the plastid; bottom-right: overlay of the fluorescent images. (B) Reconstruction of 3D images from a series of confocal fluorescence micrographs of GFP fluorescence (top), and combined GFP and chlorophyll fluorescence (bottom). GFP is primarily located in cytosolic ER strands and the nuclear envelope. P, plastid; N, nucleus. Bars, 2 µm.

View larger version (187K): [in a new window]   Fig. 4. Accumulation of GFP in the nuclear membrane (A) and on the plastid (B) surface of P. tricornutum expressing AtpC5-DDEL. Localization was demonstrated by immuno-electron microscopy using an antiserum against GFP (for details, see Materials and Methods). Arrows indicate areas that exhibit high GFP accumulation. P, plastid; N, nucleus; M, mitochondrion.

View larger version (122K): [in a new window]   Fig. 5. Accumulation of His-tagged GFP (AtpC4-His) in the central region of the plastid in P. tricornutum. (A) Top, GFP fluorescence; bottom, superimposed GFP and chlorophyll fluorescence (the red chlorophyll fluorescence marks the two lobes of the plastids, P). The fusion protein appears to accumulate between the two plastid lobes. (B) Electron micrograph of a P. tricornutum cell immuno-decorated with antisera against GFP. GFP accumulates in the central region of the plastid (arrow), where two plastid lobes are connected. Py, pyrenoid; M, mitochondrium; N, nucleus; V, vacuole. Bars, 5 µm (A); 0.2 µm (B).

View larger version (58K): [in a new window]   Fig. 6. Accumulation of GFP (AtpC6GFP) in mitochondria of P. tricornutum. (A) Confocal image of combined green GFP and red chlorophyll fluorescence. (B) 3D reconstruction of GFP and chlorophyll fluorescence of two dividing cells having two separated plastids each. Note the close association of plastids (P) and mitochondria (M). Bar, 7 µm.

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