CRC Chromosome Molecular Biology Group, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
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Abstract |
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Keywords: cyclin destruction box/green fluorescent protein/half-life/PEST sequence
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Introduction |
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The role of controlled protein degradation as a method of regulating many cellular biochemical processes has only recently been fully appreciated and the pathways involved in the accurate targetting of proteins for proteolysis are an increasingly important area of study. Failure of the key components in these pathways can be causative in human disease (Kishino et al., 1997; Matsuura et al., 1997
) and responsible for incomplete cell-cycle progression (Ghislain et al., 1993
; Finley et al., 1994
; Pu and Osmani, 1995
). Central to this process of degradation is the 26S proteosome, a 2000 kDa multi-protein complex which is distinct from the compartmentalized lysosomal proteolysis machinery and is responsible for the ATP-dependent turnover of 8090% of the cell's protein content (Lee and Goldberg, 1998
). Proteins are generally, but not exclusively, identified for systematic degradation in the proteosome by ubiquitination. The specificity of this process is in part defined by the action of a family of E2-ubiquitin conjugating enzymes, which catalyse the final ubiquitin addition, but is also conferred by sequence motifs in the target protein which act as proteolytic signals.
One such motif, the PEST sequence, is found extensively in short-lived proteins including metabolic enzymes, transcription factors and their regulators, signalling pathway components and certain cyclins (Rechsteiner and Rogers, 1996). PEST sequences are enriched for proline, glutamate, serine and threonine in a negatively or neutrally charged background and removal of this region from short-lived proteins results in more stable derivatives (Tyers et al., 1992; Pu and Osmani, 1995
; Tsurumi et al., 1995
). The metabolic enzyme ornithine decarboxylase (ODC) is a key regulatory control point in polyamine biosynthesis and is regulated by 26S proteosome degradation, albeit without prior ubiquitination, which is mediated by two PEST regions. Transfer of the carboxy-terminal ODC PEST region to a reporter enzyme, dihydrofolate reductase, resulted in an increased turnover rate of the DHFR protein (Loetscher et al., 1991
).
An alternative mechanism of targetting proteins for rapid degradation is the `cyclin destruction box' (CDB), which leads to ubiquitination and degradation of A- and B-type cyclins by the 26S proteosome at the end of mitosis. This process is promoted by the anaphase promoting complex (APC), which possesses E3-ubiquitin ligase activity, as part of its role as regulator of the metaphaseanaphase transition checkpoint. Transfer of the CDB to protein A led to destabilization of this protein in Xenopus extracts (Glotzer et al., 1991).
We wish to use destabilized GFPs to assay chromosome transmission in mammalian cells. We therefore engineered fusion proteins that link GFP to an ODC PEST sequence and a cyclin B1 destruction box region, to determine if these signals can reduce the resistance of wild-type GFP to proteolysis. The stabilities of these fusion proteins in mouse cells were compared with that of unmodified GFP.
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Materials and methods |
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All constructs were produced in a modified version of the high-level mammalian constitutive expression vector pCAGG-Zeo (Niwa et al. 1991), allowing coordinated expression of GFP and Zeocin (Invitrogen) drug resistance from a single transcript incorporating an internal ribosome entry site. The `humanized' S65T variant of GFP (Heim et al. 1995
) was cloned as a 720 bp Ecl136II/XbaI fragment into EcoRI-cut pCAGG-Zeo, after end-filling of recessed 3' ends. In the resulting plasmid, the GFP gene was flanked by reconstituted EcoRI sites, the downstream 3' site was inactivated to facilitate subsequent cloning steps. The plasmid was further modified by removal of a 760 bp region containing a polyoma virus origin of replication resulting in the 7.8 kb plasmid pCAGGGFP.
The carboxy-terminal PEST sequence corresponding to codons 423449 of the mouse ornithine decarboxylase gene (mODC) was amplified by PCR from LA-9 genomic DNA using the primers ODC-1 (5'-GAG CTG TAC AAG CAT GGC TTC CCG CCG GAG-3') and ODC-2 (5'-GAG CTG TAC ATT AAC GGT CCA TCC CGC TCT C-3') which contain BsrGI sites (underlined). A 100 bp product was isolated and cloned into the unique BsrGI site at the 3' end of the GFP gene in pCAGGGFP to produce an in-frame fusion of the PEST sequence to the carboxy terminal of the GFP protein. Sequence analysis of this plasmid, pGFPPEST revealed a single base deletion in the PCR-derived fragment at the intended TAA termination codon (in bold). This frameshift mutation resulted in the addition of a sequence of nine amino acids (NVQVKLPRL), not found in either mODC or GFP, before a TAG termination codon was reached (Figure 1A).
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Sub-cloning of the mODC PEST sequence from pGFPPEST into the BsrGI site of pCDBGFP resulted in plasmid pCDBGFPPEST in which both putative proteolytic targetting signals were incorporated in the same GFP fusion protein.
Cell culture and transfections
Mouse cell line LA-9 was maintained in DMEM supplemented with 10% fetal calf serum (Globepharm) and selection for drug-resistant lines made with 600 µg/ml Zeocin. Cell lines with stable integrations of the various GFP constructs were produced by electroporation of LA-9 cells with 10 µg of linearized plasmid DNA. Colonies were visualized and selected for GFP expression using an inverted fluorescence microscope (Leica DMIRB) with an FITC filter set (Leica I3) and several independent colonies chosen for expansion and subsequent analysis. For analysis of the CDBGFP dynamics, cells were synchronized in S phase by treatment with 2 µg/ml aphidicolin (Sigma) for 16 h. FACS analysis of GFP fluorescence was performed on a Becton-Dickinson Facscalibur machine using the supplied FITC filter set.
Metabolic labelling of proteins and immunoprecipitation
GFP half-life was determined by immunoprecipitation of pulse-labelled proteins. Approximately 1x106 cells (3040% confluent in 10 cm plates) were incubated with 50 µCi of 35S-labelled methionine (TranSLabel, ICN Pharmaceuticals) in 4 ml of methionine-deficient DMEM (Sigma) for 4 h at 37°C to pulse label all proteins including GFP. After this time, the medium was replaced with fully supplemented, unlabelled DMEM and an initial sample taken to determine the incorporation of the 35S-labelled methionine at the start of the experiment. Between five and seven further samples were taken during the next 48 h chase period at approximately 6 h time points.
For each time point, cells were lysed in 1 ml of TETN250 buffer (25 mM TrisCl pH 7.5, 5 mM EDTA, 1% Triton X-100, 250 mM NaCl, 1 mM PMSF), the soluble fraction pre-cleared with rabbit serum and GFP selectively immunoprecipitated using 1 µl of undiluted rabbit anti-GFP polyclonal antibody (IgG fraction; Clontech). GFPantibody complexes were collected using a 10% formalin-fixed Staphylococcus aureus cell suspension (Immunoprecipitin; Life Technologies) and washed with lysis buffer before separation on 12% SDSPAGE gels by standard methods (Harlow and Lane, 1988). Gels were dried under vacuum after fixing in methanolacetic acidglycerol and electronic autoradiography was carried out using an Instant Imager (Packard Bell Instruments). The total radioactivity in bands corresponding to GFP or its tagged variants was calculated by volume integration using the built-in software after background correction. The GFP half-life was calculated by linear regression analysis of log(total radioactivity per band) against time.
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Results |
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Immunoprecipitated samples of pulse-chase labelled protein extracts were taken over a 48 h period to determine the stabilities of each of the fusion proteins (Figure 2). As predicted from the structure, CAGGGFP was a stable protein whose destruction followed first-order kinetics with a half-life of approximately 26 h. The fusion protein containing the C-terminal mouse ODC PEST sequence was also degraded with first-order kinetics, but at an increased rate, resulting in a reduced half-life of 9.8 h. This represents a significant destabilization of the protein of 2.6-fold compared to CAGGGFP.
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The addition of the PEST motif to this CDBGFP protein marginally reduced the average half-life further to 5.5 h. The outstanding feature of this latter `combination' fusion is that overall levels of fluorescence were the lowest for any of the fusions, such that no labelled protein could be detected on the final time point sample. Also, FACS analysis of unsynchronized cells expressing CDBGFPPEST showed a bimodal distribution of fluorescence where the lower peak coincided with the non-fluorescent negative control (data not shown). Thus, addition of the PEST region does not appear to make a significant difference to fluorescence when protein levels are non-limiting; at lower concentrations, its effect is more evident.
The most prominent consequence of fusing the GFP sequence to the cyclin destruction box was highlighted through a time-dependent analysis of fluorescence development and decay in cells from synchronized culture (Figure 3A and F). Cells from lines containing either the CAGGGFP or CDBGFP variants were blocked in S phase using the DNA synthesis inhibitor aphidicolin (Sourlingas and Sekeri-Pataryas, 1996
). The FACS profiles of these cells immediately after removal of the synthesis block showed similar distributions of fluorescence intensities, with wild-type GFP being slightly higher (Figure 3C and E
). However, over a period of 26 h, corresponding to approximately one and a half cell cycles for LA-9 cells, the population profiles differed dramatically. In order to measure the early stages of fluorescence loss, observed as an initial moderate shift in the FACS profile peak to the left in Figure 3D
, cells were defined as fluorescent if they exhibited a relative fluorescence intensity of
200 units. Inevitably, below this threshold some cells were included as non-fluorescent despite expressing significant levels of GFP. By this criterion, the percentage of fluorescent cells remained constant (8085%) in the line expressing CAGGGFP, whereas the CDBGFP line showed a loss of fluorescence down to 30% as the synchronized cells entered mitosis. Fluorescence levels increased subsequently as cells progressed through G1 and S phase before a second decrease occurred as cells entered their second mitotic division. The reduced peak in fluorescence levels during this second cycle (62% compared with 78%) results from the loss of synchronization in the population after this length of time. These dynamic changes in CDBGFP fluorescence with cell cycle progression are illustrated in Figure 3F
, in which each cell of a synchronized pair undergoes mitosis resulting in reductions in fluorescence levels. At the four-cell stage, once the cells are in interphase, fluorescence levels have returned to a maximum and are comparable between all cells. Although it is not possible from these data to establish the exact timing of these changes with respect to the cell cycle, the pattern is consistent with that reported for normal cyclin B1 accumulation and degradation (Glotzer et al., 1991
).
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Discussion |
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The reduction in GFP stability seen in the mODC PEST-tagged variant, while significant, still places GFP at the higher end of stability compared with more traditional reporters of gene activity such as luciferase which has a half-life of approximately 3 h (Thompson et al., 1991). In addition, the lack of enzymatic amplification of a signal with GFP means that its sensitivity is limited, as cytoplasmic concentrations of approximately 1 µM are required to distinguish signal from auto-fluorescent background (Niswender et al., 1995
). However, as most enzymatic methods involve cell disruption, this improvement in using GFP non-invasively with a shorter half-life will be suitable for many applications. In particular, for monitoring chromosome transmission, where expression of GFP to readily detectable levels is not problematic, the shorter half-life means that parentally derived cytoplasmic GFP is removed quickly, allowing the measurement of de novo GFP synthesis from the daughter cells.
GFP may yet be a viable alternative for in vivo gene expression studies if the destabilization can be improved to reduce the half-life further. A destabilized GFP variant, d2GFP, has recently been described (Li et al., 1998) which utilizes a PEST region very similar to that used in this work. However, the reported half-life of this variant is 2 h and further reports of a 1 h variant (d1GFP) are based on fluorescence measurements, not biochemical purification. The discrepancy between those claims and the results presented here has been investigated to distinguish between cell differences, experimental protocols and intrinsic properties of the GFP variants used. We have confirmed the increased rate of degradation of the d1GFP variant, compared with the GFPPEST described here (data not shown), and using the more accurate method of pulse-chase labelling we have determined its half-life to be 50 min in human HT1080 cells.
This result is remarkable considering the overall homology between the two mODC PEST sequences used. The d2GFP variant is extended compared with GFPPEST by only 13 amino acids, but the core PEST region is maintained between both. This clearly implicates `non-PEST motif' residues as having a major contributory role to the rapid turnover of the mouse ornithine decarboxlase protein ordinarily. What is more significant, however, is that in the most unstable d1GFP variant the conserved Glu residues in the PEST region are mutated to Ala. While these changes would theoretically predict a weaker PEST motif, in practice the alterations result in a 50% reduction in protein stability. Consequently, the presence of a PEST motif within a protein merely implies a propensity to instability and cannot indicate the magnitude of the degradation rate even when close to or identical with the consensus PEST sequence.
While the patterns of protein degradation for the cyclinGFP fusion proteins are more complex than the simple linear profiles obtained with the PEST fusions, these variants are potentially useful as non-invasive indicators of cell-cycle status. The FACS profile of unsynchronized cells expressing these proteins (Figure 3D) is an indirect measurement of the proportion of cells in defined stages of the cell cycle. Although more detailed investigation of the correlation between fluorescence levels and cellular status is necessary, the lower peak of the fluorescence distribution corresponded broadly to mitotic and early G1 cells. Therefore, this form of GFP could be used as an alternative to procedures, such as propidium iodide staining, which require fixation with concomitant cell death. Indeed, in the cell synchronization experiments described (Figure 3AE
), the fluorescence levels were a direct indication of the efficiency of the aphidicolin treatment.
However, it is still debatable as to how accurately GFP fluorescence intensity is a reliable indicator of GFP levels in a cell. Two factors that significantly affect the fluorescent properties of GFP, namely the requirement for post-translational oxidative fluorophore formation and the sensitivity to cellular pH, could result in fluorescence intensities being lower than actual GFP concentration. Western analysis indicates that a good correlation exists between fluorescence intensity and protein concentration (Li et al., 1998), but does not distinguish between immature, non-fluorescent and mature, fluorescent forms of GFP.
Considering this correlation between fluorescence levels and protein content, it is paradoxical that cell lines containing the unstable GFPPEST construct (half-life 9.8 h) have fluorescence intensities not significantly different to those of lines containing its more stable GFP counterpart (half-life 26 h). Differences in steady-state protein levels between these lines would predict that the more unstable lines are also the least intense for GFP fluorescence, contrary to the actual observation. A similar observation was reported for the d2GFP variant (Li et al., 1998), although in our experimental system lines containing this construct evidently displayed weaker fluorescence both in FACS analysis and by microscopy. Although this could indicate that non-fluorescent immature GFP molecules are preferentially degraded, we believe this to be unlikely. Instead, it is probable that proteolytic products from 26S proteosome activity maintain a degree of fluorescence (Tsien, 1998
) but are not detected by the immunoblot technique used to measure half-life, thereby increasing the apparent GFP concentration. Thus, reduction in fluorescence intensity is proportional to a decrease in GFP half-life, but not to the same magnitude. The d1GFP variant that is 30 times more unstable than the wild-type GFP is, nonetheless, only ~7080% less intense by FACS analysis.
The results also shed some light on the biology of proteolytic signalling itself. The GFPPEST fusion construct has been tested in human HT1080 and HeLa-S3 cell lines where the fusion protein localized to give a punctate pattern. The distribution of GFP foci appeared limited to the cytoplasm, partially concentrated around the nuclear envelope (data not shown). This phenotype was only seen in LA-9 cells when the brighter EGFP variant was fused to mouse ODC PEST, although the phenotype was less pronounced than in human cells. This mottled pattern is unlikely to be due to sub-cellular localization of the 26S proteosome, which is known to be found in both the nucleus and cytoplasm (Coux et al., 1996), but may represent natural sequestering of proteins marked for degradation away from the active cellular protein conterparts. Equally, the phenotype may be artefactual, with the very high levels of expression from the pCAGG vector resulting in protein aggregation complexes.
The distribution of the CDBGFP protein was also consistent with known cyclin B1 characteristics (Hagting et al., 1998). A pronounced nuclear localization was observed in mouse and human HT1080 cells (Figure 3F
, 0 h, lower panel). Nuclear-cytoplasmic translocation is a normal feature of cyclin B1 expression and is mediated by a putative cytoplasmic retention or nuclear export signal between residues 129 and 157 (Hagting et al., 1998
; Toyoshima et al., 1998
) which is absent from the CDBGFP construct (Figure 1A
). In this respect, CDBGFP is a useful starting point for the in vivo analysis of the 5' region of the gene which is responsible for the dynamic movement of the cyclin B1 protein throughout the cell cycle.
In conclusion, it is evident from the results using the PEST and CDB fusions that the barrel-like structure of GFP is not refractory to major modifications that alter the basic properties of GFP biology. In these examples, GFP stability can be permanently compromised by the addition of the appropriate proteolytic signal sequences. Engineering opportunities are, however, limited to the carboxy- and amino-terminal ends for GFP, as internal modifications destroy the protective barrel structure and expose the fluorophore rendering it non-fluorescent (Dopf and Horiagon, 1996). However, the ease by which new derivatives can be produced and tested directly in living cells will allow GFP to be tailored to a variety of individual uses.
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Acknowledgments |
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Notes |
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References |
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Received December 11, 1998; revised August 26, 1999; accepted September 1, 1999.