Figure 1.
Chlamydomonas transformation vectors for protein targeting to specific subcellular locations.
A–D. Schematic representation of Chlamydomonas targeting vectors. All transformation vectors contain the hsp70/rbcs2 promoter (P), the ble gene that confers resistance to zeocin, the 2A self-cleaving sequence from foot-and-mouth-disease virus, and the rbcs2 terminator (T). The site of cleavage is indicated with an arrowhead. A. pBR28, mCerulean is targeted to the nucleus by a C-terminal fusion to 2xSV40 NLS. B. pBR29, mCherry is targeted to the mitochondria by an N-terminal fusion to the mitochondrial transit sequence (MTS) of mitochondrial atpA. C. pBR32, mCherry is targeted to the chloroplast using the chloroplast transit sequence (CTS) from psaD. D. pBR30/31, mCherry is targeted to the ER using the ER-transit sequence (ER-TS) from either BiP1 or ars1. The ER retention sequence H-D-E-L is fused to the C-terminus of mCherry. E–P. Microscopy images of cells transformed with pBR28 (E–G), pBR29 (H–J), pBR32 (K–M), and pBR30 (N–P). Top row are live cell images of the fluorescent proteins targeted to the nucleus (E), mitochondria (H), chloroplast (K) or ER (N, O). (I) The cell is co-stained with the mitochondrial dye Mitotracker. (N) Cross section through the top of a cell expressing mCherry in the ER allows for the visualization of the cortical ER network. (O) Cross section through the middle of the same cell as in (N). The chloroplast membranes are visualized in (F), (L) and (P). Merged images are shown in the bottom row.
Table 1.
Summary of transit peptides and targeting sequences used in this study.
Figure 2.
Gene stacking using a multi-cistronic transformation vector.
A. A schematic representation of the Chlamydomonas multi-cistronic expression vector. The expression of the cassette is under the control of the hsp70/rbcs2 promoter (P). Ble confers zeocin-resistance. mCerulean is targeted to the nucleus with the SV40-NLS. mCherry is targeted to the ER using the BiP ER-TS and the HDEL retention sequence. Two 2A self-cleaving sequences are fused between the three cistrons: F2A, from FMDV1; E2A, from equine rhinitis A virus. Black arrows represent the location of the oligonucleotides used in (B). Following co-translational processing of the 2A peptides, three distinct proteins are expressed (ovals). B. PCR analysis of the multi-cistron cassette genome integration. Transformants were screened by PCR to identify individual clones that correctly integrated the multi-cistronic transformation vector, using the oligonucleotides indicated by the arrows in (A). Three independent clones (cl) are shown. L, ladder; WT, wildtype cc1690; NTC, no template control; P, plasmid. C. Live cell fluorescence microscopy of a clone expressing the multi-cistronic vector. mCerulean-NLS (blue) localizes to the nucleus while BiP-mCherry-HDEL (red) is targeted to the ER. Scale bar, 5 μm.
Figure 3.
An mt+ strain transformed with pBR30 (A) was crossed with an mt- strain transformed with pBR28 (B). Progeny that expressed mCherry in the ER and mCerulean in the nucleus were obtained (C). Cell lines expressing both ER-mCherry and nuclear mCerulean were crossed with cells transformed with mitochondria-targeted Venus (D), to obtain progeny that stably expressed three distinct FPs in three sub-cellular locations (E). These cell lines were crossed to transgenic cells that expressed α-tubulin (TUA1) fused to mTagBFP (F), to obtain progeny that expressed four FPs in four distinct subcellular locations (G). H. Fluorescence plate reader assays of the parents and progeny indicate that FP expression remains stable following matings. Cell lines were assayed for mCherry expression (ex575/em608), mCerulean expression (ex450/em486), and Venus expression (ex515/em550). ER-mCherry parents (ER-mCher 10 and 27), nuclear mCerulean parent (Cerulean NLS) and mitochondrial Venus parents (MTS Venus 19 and 28) are shown along with WT cc1690. 2 color cell lines 1–4 express ER-mCherry and Cerulean-NLS. 3 color cell lines 1–6 express ER-mCherry, nuclear mCerulean and mitochondrial Venus. Scale bars, 5 μm.