Plant Physiol, February 2002, Vol. 128, pp. 341-344

SCIENTIFIC CORRESPONDENCE

When a Day Makes a Difference. Interpreting Data from Endoplasmic Reticulum-Targeted Green Fluorescent Protein Fusions in Cells Grown in Suspension Culture¹

North Carolina State University, Department of Botany, Raleigh, North Carolina 27695-7612

	ARTICLE

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The stability of the self-contained structure of green fluorescent protein (GFP) has made it the most widely utilized fluorescent marker for gene expression and subcellular localization studies (Chalfie et al., 1994; Tsien, 1998; De Giorgi et al., 1999; Haseloff et al., 1999). This same stability, however, may contribute to misinterpretation of the fluorescence images. Using a constitutively expressed endoplasmic reticulum (ER)-targeted GFP fusion peptide, we found that although there was no change in the GFP fluorescence in the transformed tobacco (Nicotiana tabacum) cells, the GFP fusion peptide was degraded over time. Most importantly, fluorescence microscopy alone was not an effective means for monitoring the presence of the full-length fusion peptide in this system.

The major advantages of using GFP are that the fluorescence is maintained even when fused to peptide fragments or intact proteins, that GFP fusion proteins can be readily monitored directly in living cells and tissues, and that GFP causes little perturbation to the polypeptide to which it is fused (Leffel et al., 1997; Tsien, 1998; De Giorgi et al., 1999; Margolin, 2000). Researchers designing GFP-fusion proteins routinely check for functional activity of the recombinant fusion peptide in vitro and use GFP fluorescence as a convenient indicator for the presence of the recombinant peptide in vivo. In addition, monitoring GFP fluorescence in vivo has proven to be an effective mechanism for monitoring protein turnover (Cronin and Hampton, 1999; Silverstone et al., 2001). While this is a valid approach when GFP is fused to a full-length protein that is ubiquitinated and subsequently completely degraded (e.g. ER-localized 3-hydroxy-3-methyl glutaryl-CoA reductase; Ravid et al., 2000), it would not be valid if GFP or a peptide fragment containing GFP were cleaved from the fusion peptide before the peptide was targeted for degradation. Our experience is that fragmentation of recombinant polypeptides from an ER-targeted GFP-fusion polypeptide can occur without a detectable loss of in vivo fluorescence in the tobacco cells and that the extent of loss of the recombinant polypeptide correlates positively with cell growth rate and time in culture. Therefore, when studying cells grown in suspension culture, in vivo fluorescence is not a sufficient indicator of the full-length GFP-fusion peptide.

In studies of ER-calcium homeostasis of tobacco cells grown in suspension culture, we used a fusion peptide that consists of an ER-targeted GFP fused to the carboxy-terminal (C)-domain of calreticulin (CRT). Specifically, tobacco suspension culture (NT1) cells were transformed with binary plasmids carrying either an ER-targeted mgfp5 minus the ER retention sequence (Haseloff et al., 1997) fused in frame with the last 366 nucleotides in the maize (Zea mays) cDNA sequence of CRT (corresponding to 122 amino acids of the C-domain of maize CRT including the HDEL sequence), or a full-length ER-targeted mgfp5 alone (Fig. 1). All constructs were under the control of a 35S promoter, and kanamycin resistance was used as the selectable marker. Ten independent, kanamycin-resistant cell cultures were isolated for each transgene. Four cell lines transformed with the GFP:C-domain fusion construct (denoted GCd-A, GCd-E, GCd-I, and GCd-J) and one control cell line transformed with ER-targeted mgfp5 (denoted GFP) were selected. All of the cell lines were imaged using a laser scanning confocal microscope, and similar expression patterns and cell morphologies were observed.

View larger version (26K): [in this window] [in a new window]   Figure 1.   Plasmid constructs. Both constructs used a CaMV35S promoter and octopine synthase (OCS) terminator. pGFP contained the mgfp5 sequence that contains a 5' signal sequence (SS) derived from a gene encoding the ER-localized chitinase and an HDEL ER retention sequence as noted (Haseloff et al., 1997). pGFP:C-domain contained mgfp5 with the HDEL sequence removed fused upstream of the C-domain (366 nucleotides of the 5' end) of the CRT open reading frame that included the HDEL ER-retention sequence as noted. Numbers in parentheses indicate nucleotide position.

Figure 2A shows the growth curves for the wild-type and the transgenic lines. Both GCd-A and GCd-E displayed a significant lag in growth, and they reached the early stationary growth phase 1 and 2 d later, respectively, than the wild-type and the GFP control. The delayed growth rate of GCd-A and GCd-E lines also was observed with the two other GFP:C-domain lines (GCd-I and GCd-J). To investigate how the recombinant GFP:C-domain fusion peptide was affected by cell growth, microsomes from 3-, 4-, and 5-d-old cells were isolated and proteins separated by SDS-PAGE, blotted, and immunostained with polyclonal antibodies against either GFP (Clontech, Palo Alto, CA) (1:3,000, Fig. 2B) or a maize CRT (1:5,000, Fig. 2C). Typical expression patterns of wild-type and the transgenic lines for d 3, 4, and 5 are shown. ER localization of the GFP:C-domain fusion peptide was confirmed by Suc gradient analysis as previously described (Persson et al., 2001).

View larger version (33K): [in this window] [in a new window]   Figure 2.   Increased degradation of GFP:C-domain fusion over time in NT1 suspension culture cells. A, Fresh weight of NT1 cells from wild-type, GFP:C-domain lines GCd-A and GCd-E and GFP-control (GFP) over time. Values plotted are the averages of at least seven samples (SEs are indicated). Lines are drawn to show the best fit curve to the average fresh weight. B and C, Microsomes were isolated from cell lines wild-type (WT), GFP:C-domain transgenic lines (GCd-A and GCd-E), and an ER-targeted transgenic mgfp5 control line (GFP) on 3, 4, and 5 d after transfer as noted. Equal amounts of microsomal proteins were analyzed by 10% SDS-PAGE (10 µg of protein/lane), blotted, and immunostained with polyclonal antibodies against GFP (1:3,000) (B), or with polyclonal antibodies against maize CRT (1:5,000) (C). D, Confocal fluorescence microscopy of NT1 cell lines. Confocal fluorescence images were taken of cells from ER-targeted transgenic GFP-control line (ER-GFP) and GFP:C-domain transgenic lines GCd-A and GCd-E, from d 3 to 6 after transfer to new NT1 culture medium. GFP was excited at 488 nm using an argon laser. Each image is a projection of 32 confocal planes. The GFP fluorescence emission was recorded from 500 to 550 nm. Simultaneous differential interference contrast images were recorded using a transmitted light detector (Scale bar = 25 µm).

A comparison of two cell lines with slightly different growth rates emphasized the effect of growth rate on the stability of the recombinant ER-fusion protein. As shown in Figure 2B, line GCd-A had more of the lower M_r GFP-cross-reacting bands (35- and 27-kD bands) than line GCd-E. By d 5, the size of the major breakdown product correlated with a 27-kD peptide that was recognized by a polyclonal antibody against GFP, but not by the polyclonal antibody against maize CRT (compare Fig. 2B and 2C). These data indicate that the CRT C-domain polypeptide was lost. Endogenous CRT (Fig. 2C) and immunoglobulin binding protein (data not shown) showed no signs of degradation within the time frame of these studies, which suggested that there was not a general loss of endogenous proteins in the ER of the transformed NT1 cells. The loss of the GFP:C-domain fusion protein did not arise from an increase in dead cells in culture, as there was no significant difference in cell viability monitored with fluorescein diacetate over the time course of these studies.

The redesigned mgfp5 used to construct the fusion peptide had the HDEL sequence removed so that both the residual 35- and 27-kD peptides could have been trafficked to the vacuole or secreted by the cells. Analysis of the distribution of the recombinant peptides in microsomes separated on discontinuous Suc gradients revealed that the 35-kD peptide, like the full-length fusion protein, localized primarily with an ER-enriched fraction. Some of the 27-kD GFP peptide was recovered in the ER-enriched fraction; however, most was recovered on the top of the gradient, suggesting that it was preferentially trafficked to the vacuole or more readily released from membrane vesicles upon homogenization. Importantly, both the 27- and the 35-kD GFP-cross-reacting peptides fluoresced when the blots were analyzed with a Storm 860 Imaging System (Amersham Biosciences Inc., Piscataway, NJ; 450-nm excitation and 520-nm emission) indicating that both peptides could potentially contribute to the in vivo fluorescence.

To assess whether cells in which there was a significant loss of the GFP:C-domain fusion peptide displayed any variations in fluorescent patterns or intensity, cells from the GFP:C-domain fusion lines and GFP-control line were imaged with a Leica DMIRBE fluorescence microscope equipped with a Leica PL APO 140× NA 1.25 oil immersion lens and coupled to a Leica TCS 5P laser scanning head (Leica, Wetzlar, Germany). GFP was excited at 488 nm using an argon laser, and GFP fluorescence emission was recorded from 500 to 550 nm. As shown in Figure 1D, the in vivo fluorescence was reticular, consistent with ER localization, and there was no significant difference in the fluorescence pattern between cells from d 3 to 5. If the 27-kD GFP peptide was localized in the vacuole or cell wall, the low pH in these regions may have quenched the fluorescence and decreased sensitivity; therefore, the fluorescence may not have been detected (Scott et al., 1999).

Consistently, the rate of degradation of the GFP:C-domain fusion peptide correlated positively with the growth of the cells (Figs. 2A and 3B). Most importantly, even when line GCd-A contained 4 to 5 times more of the 35- or 27-kD GFP peptides than line GCd-E (Figs. 2B and 3B), the in vivo fluorescence images were indistinguishable (Figs. 2D and 3A), indicating that the observations of in vivo ER fluorescence were not sufficient to conclude that the full-length GFP:C-domain fusion peptide was still present in this system.

View larger version (76K): [in this window] [in a new window]   Figure 3.   Degradation of the GFP:C-domain fusion protein in late stages of growth for GFP:C-domain lines GCd-A and GCd-E. A, Confocal fluorescence images were taken of cells from the GFP:C-domain transgenic lines GCd-A and GCd-E and the ER-targeted mgfp5 control line (ER-GFP) from late stages of the culture period as noted (Scale bar = 10 µm). B, Microsomal proteins from the same cell lines were analyzed by 10% SDS-PAGE (10 µg of protein/lane), blotted, and immunostained with polyclonal antibodies against GFP (1:3,000). Migrations of GFP and the full-length GFP:C-domain fusion peptide are indicated. The GFP:C-domain peptide and the proteolytic products indicated, which cross-reacted with GFP antibodies, also fluoresced when analyzed with a Storm 860 Imaging system.

In summary, the transformed NT1 cells exhibited similar fluorescent patterns and intensities even when a significant amount of the ER-targeted GFP:C-domain fusion peptide was degraded. With the increased use of GFP-fusion peptides for subcellular localization of proteins, care must be taken to use cells growing at the same rate and to constantly perform immunoblot analysis of cellular proteins to monitor the expressed GFP fusion peptides. Importantly, when using cells growing in suspension culture, a day can make a difference in the interpretation of the data.

	ACKNOWLEDGMENTS

The maize CRT cDNA was kindly provided by Dr. Rebecca S. Boston (North Carolina State University, Raleigh). Antisera against maize CRT was kindly provided by Dr. Jeffrey Gillikin and Dr. Rebecca S. Boston (North Carolina State University). We thank Jonathan Swaffield (North Carolina State University) for his helpful discussions on proteolysis. The confocal imaging was performed at the North Carolina State University Cell and Molecular Imaging Center under the direction of Dr. Nina Strömgren Allen. Upon request, all novel materials described in this publication will be made available without restrictions for non-commercial research purposes.

	FOOTNOTES

Received September 12, 2001; returned for revision October 7, 2001; accepted November 4, 2001.

¹ This work was supported in part by the National Aeronautics and Space Administration (grant no. NAGW-4984) and in part by funding from the North Carolina Agricultural Research Service (to D.R., W.F.T., and W.F.B.).

² These authors have contributed equally to presented work.

^* Corresponding author; e-mail wendy_boss{at}nscu.edu; fax 919-515-3436.

www.plantphysiol.org/cgi/doi/10.1104/pp.010840.

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