Versatile cassette design and implementation
To differentiate the pivotal roles of ER resident proteins requires the design of functional probes that facilitate protein localization and interactions in an oxidized environment. Engineered FPs provide a diverse array of tools for biological imaging [43-46] and encompass techniques that evaluate protein dynamics [e.g. fluorescence recovery after photobleaching (FRAP) [47-50]], protein-protein interactions [e.g. fluorescence resonance energy transfer (FRET) [51-54]], and organelle continuity [e.g. fluorescence loss in photobleaching (FLIP) ]. In contrast, the Strep-tag II affinity tag  coupled with effective procedures for detection, purification and immobilization has resulted in the rapid isolation of recombinant gene products . In this study, conventional FPs and affinity tags were modified and select ER chaperones and foldases that assist in protein maturation, degradation and export (Figure S1, Table 1) were evaluated.
To promote the utility of our PCR-based modules (Figure 1, Tables S1 and S2), cassettes were designed with several considerations. First, to identify protein complexes localized to the early secretory pathway, affinity and FP tags have been employed with native ER retrieval motifs – either an HDEL or KDEL sequence. Second, although GFP and DsRed variants result in well-separated spectra for multicolor imaging, an in-depth evaluation of FP variants in the ER lumen of S. cerevisiae has not been investigated . Previous studies suggested that exposed cysteines (e.g. C49 and C71) within the ER lumen may bind maturing GFP molecules or unfolded proteins containing cysteines, thus resulting in mixed disulfides. To prevent dimerization of fluorescent probes in vivo, monomeric versions of GFP variants utilize the A206R mutation . Consequently, yEmCFP and yEmCitrine  were incorporated in the pCY H/KDEL cassettes. Conversely, mCherry is a derivative of DsRed that contains no exposed cysteine residues . DsRed variants do fluoresce effectively in an oxidized environment – specifically the periplasm of Escherichia coli . In this study, fluorophores for four-color imaging have been codon-optimized for yeast . With respect to cassette design, the introduction of a flexible linker between a target protein and tag is particularly advantageous, resulting in improved function  and purification . The implementation of a yeast-enhanced or codon-optimized linker encoding GFP variants resulted in a twofold increase in fluorescent intensity in previous studies . Consequently, the pCY H/KDEL cassettes contain a yeast-enhanced polylinker, as well as FP variants and affinity tags codon-optimized for yeast.
Figure 1. Depiction of key components in pCY H/KDEL cassettes, adapted from Young and colleagues (77). A) Plasmid map of the template used for this cassette series, which contains C-terminal ER-retrieval sequences (denoted by a five-point star). B) Generalized nucleotide sequence of all pCY H/KDEL cassettes, similar to modules derived from pFA6a-GFP(S65T) . Nucleotides corresponding to retrieval sequences, FEHDEL and MLKDEL, are identical to the final six amino acids of BiP/Kar2p and Scj1p, respectively, in Saccharomyces cerevisiae. To amplify specific tags and selection markers (SM), regions of primer homology are denoted in italics. Restriction sites have been identified, and corresponding nucleotides underlined. A polylinker was designed based on frequently-used codons of Saccharomyces cerevisiae  as denoted by frequency (i.e. percentage of codon usage within the entire genome) and inserted between the XmaI and original PacI restriction site, thereby eliminating its uniqueness. The ADH1 terminator (TADH1) of S. cerevisiae is a component of all pCY H/KDEL plasmids that are derivatives of parental vectors consisting of kanMX. C) Arrangement of all blue, green, yellow, and red fluorescent protein variants of the pCY H/KDEL series. D) Epitope tags, c-myc and HA, contain codon-optimized [i.e. yeast-enhanced (yE)] sequences. Residues that maintain affinity for commercial antibodies are in bold. E) A modified streptavidin tag, Strep-tag II, was constructed with an N-terminal thrombin cleavage site and C-terminal HDEL retrieval sequence. The recognized cleavage site is denoted by (^). As a consequence of thrombin's enzymatic activity, remaining amino acids are underlined. The Strep-tag II epitope is shown as bold font. F) General design for forward and reverse primers using standard homologous recombination techniques.
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Experimental design and assessment of H/KDEL modules
The pCY H/KDEL cassette series promotes the facile modification of ER folding factors (Figure S1). Proteins whose modifications have been reported to compromise cell growth and activate cellular stress responses [e.g. UPR and heat shock response (HSR)] were considered ideal targets. A significant percentage of these proteins are characterized as essential (i.e. critical for cell survival) and are intimately associated with ER quality control (ERQC) (Table 1). However, their respective functions are quite diverse: ER translocation, protein folding and maturation, ERAD, COP II vesicle budding (i.e. trafficking from ER-to-Golgi, defined as anterograde transport), retrograde transport (i.e. trafficking from Golgi-to-ER), and N- and O-linked glycosylation.
ER resident proteins are generally acknowledged to be upregulated following UPR induction . As summarized in Table 1, the UPR regulates genes of the early secretory pathway to varying degrees. Of the six canonical UPR genes – KAR2, EUG1, PDI1, LHS1, FKB2 and ERO1 [4, 63, 64] – the effects of ER resident proteins, BiP/Kar2p and Pdi1p, fused to FP variants and affinity tags were evaluated in this work.
Commonly referred to as BiP, Kar2p is the yeast homolog of binding protein immunoglobulin BiP [also known as Grp78 ]. As one of two distinct Hsp70 molecular chaperones of the ER lumen, BiP is conserved throughout eukaryotes and binds preferentially to hydrophobic residues of nascent or unfolded proteins [66, 67]. Although a resident of the ER lumen, S. cerevisiae BiP/Kar2p traffics to the cis-Golgi where it is retrieved due to its C-terminal HDEL tetrapeptide and direct association with Erd2 [68, 69]. Endogenous BiP is essential for cell growth and strain viability  and maintains a molecular weight of 74.5 kDa. Similarly, protein disulfide isomerase (Pdi1p) is a ER resident protein conserved in all eukaryotes, required for cell growth , and contains an HDEL retrieval motif. Pdi1p functions as a catalyst by promoting the formation, reduction and isomerization of disulfide bonds in nascent proteins [72, 73], and maintains a molecular weight of 58.2 kDa. These attributes provide a unique experimental system to evaluate recombinant BiP and Pdi1p expressed under the regulation of their native promoter in S. cerevisiae.
Our research interests focus on the spatiotemporal dynamics of ERQC and protein trafficking within the early secretory pathway; thus, we investigated the effects of FPs variants and affinity tags fused to ER resident proteins – BiP, Pdi1p and Scj1p – in multiple strains. Saccharomyces cerevisiae strains BJ5464 [MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL, ATCC® 208288™, ], BY4742 [MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0, EUROSCARF Y1000, ], W303 [MATα ade2-1 ura3-1 his3-11 trp1-1 leu2-3 leu2-112 can1-100, ATCC® 208353™, ] and derivatives listed in Table 2 were used in this study. Yeast strain BJ5464 lacks efficient vacuolar degradation due to the deletion of PEP4 and PRB1 and is predominantly used for heterologous protein expression. In contrast, BY4742 is typically used to evaluate endogenous cellular processes and protein function, while numerous studies have examined UPR initiation and attenuation in W303.
Table 2. Saccharomyces cerevisiae strains used in this study
|BJ5464||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL||—||ATCC 208288™/|
|CY2007||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL KAR2-Cerulean-HDEL::hphMX4||HRF1 KAR2, HRR KAR2||This study|
|CY2008||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL KAR2-yEpolylinker-yEmCFP-HDEL::hphMX4||HRF2 KAR2, HRR KAR2||This study|
|CY2009||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL KAR2-Venus-HDEL::kanMX||HRF1 KAR2, HRR KAR2||This study|
|CY2010||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL KAR2-yEpolylinker-yEVenus-HDEL::kanMX||HRF2 KAR2, HRR KAR2||This study|
|CY2011||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL KAR2-yEpolylinker-yECitrine-HDEL::kanMX||HRF2 KAR2, HRR KAR2||This study|
|CY2012||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL KAR2-yEpolylinker-yEmCitrine-HDEL::kanMX||HRF2 KAR2, HRR KAR2||This study|
|CY2013||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL KAR2-yEpolylinker-yEGFP-HDEL::kanMX||HRF2 KAR2, HRR KAR2||This study|
|CY2014||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL KAR2-mCherry-HDEL::hphMX4||HRF1 KAR2, HRR KAR2||This study|
|CY2015||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL PDI1-yEpolylinker-yECFP-HDEL::hphMX4||HRF2 PDI1, HRR PDI1||This study|
|CY2016||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL PDI1-yEpolylinker-yEmCFP-HDEL::hphMX4||HRF2 PDI1, HRR PDI1||This study|
|CY2017||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL PDI1-Venus-HDEL::kanMX||HRF1 PDI1, HRR PDI1||This study|
|CY2018||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL PDI1-yEpolylinker-yEVenus-HDEL::kanMX||HRF2 PDI1, HRR PDI1||This study|
|CY2019||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL PDI1-yEpolylinker-yEGFP-HDEL::kanMX||HRF2 PDI1, HRR PDI1||This study|
|CY2020||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL PDI1-mCherry-HDEL::hphMX4||HRF1 PDI1, HRR PDI1||This study|
|CY2021||MATα ura3-52 trp1 leu2Δ his3Δ200 pep4::HIS3 prb1-Δ1.6R can1 GAL SCJ1-Venus-KDEL::kanMX||HRF1 SCJ1, HRR SCJ1||This study|
|W303||MATα ade2-1 ura3-1 his3-11 trp1-1 leu2-3, 112 can1-100||—||ATCC® 208353™/|
|CY4001||MATα ade2-1 ura3-1 his3-11 trp1-1 leu2-3, 112 can1-100 KAR2-mCherry-HDEL::hphMX4||HRF1 KAR2, HRR KAR2||This study|
|CY4002||MATα ade2-1 ura3-1 his3-11 trp1-1 leu2-3, 112 can1-100 SCJ1-Venus-KDEL::kanMX||HRF1 SCJ1, HRR SCJ1||This study|
|BY4742||MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0||---||EUROSCARF Y1000/|
|CY6006||MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 KAR2-yEpolylinker-Cerulean-HDEL::hphMX4||HRF2 KAR2, HRR KAR2||This study|
|CY6007||MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 KAR2-yEpolylinker-yEGFP-HDEL::kanMX||HRF2 KAR2, HRR KAR2||This study|
|CY6008||MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 KAR2-mCherry-HDEL::hphMX4||HRF1 KAR2, HRR KAR2||This study|
|CY6009||MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 PDI1-yEpolylinker-yEGFP-HDEL::kanMX||HRF2 PDI1, HRR PDI1||This study|
|CY6010||MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 PDI1-mCherry-HDEL::hphMX4||HRF1 PDI1, HRR PDI1||This study|
|CY6011||MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 SCJ1-yEpolylinker-yEmCitrine-KDEL::kanMX||HRF2 SCJ1, HRR SCJ1||This study|
Implementation of the HDEL retrieval sequence: expression, ER localization and trafficking effects
The expression of BiP-FP fusions – incorporating blue, green, yellow and red variants designed with the C-terminal HDEL retrieval sequence – was assessed by a facile and direct approach. As previously described , an in-gel fluorescent technique enabled the discrimination of the intrinsic fluorescence of FP variants when fused to the ER translocon, Sec61, a membrane protein whose C-terminus is exposed to the cytosol. We exploited the spectral characteristics of FP variants expressed in the oxidized environment of the ER in order to examine BiP fusions whose excitation and emission profiles constitute the entire spectrum of visible light. As shown in Figure 2A, in-gel fluorescent methods also provide an adequate means of examining BiP-FP fusions, as compared to parental strains.
Figure 2. Expression of BiP/Kar2p fluorescent protein fusions implementing the HDEL retrieval sequence. A) In-gel fluorescence analysis of GFP and DsRed variants fused to an ER resident protein, BiP, compared to parental strains. FP fusions were detected at excitation wavelengths of 457, 488, or 532 nm. B) Immunoblotting of the Hsp70 chaperone, BiP. Western blot analysis shows corresponding lanes analogous to (A); protein bands were probed with anti-Kar2p and compared to total protein load via an appropriate control, anti-Actin. C) Comparison of full-length BJ5464 BiP-FP fusions via in-gel fluorescence analysis under non-reducing conditions. FP fusions were detected at excitation wavelengths of 457, 488, or 532 nm. Oligomers and mixed disulfides were not detected by intrinsic fluorescence of the variants. D) Western blot analyses confirmed the formation of one species under non-reducing conditions. Endogenous BiP (WT) was probed by anti-Kar2p and BiP-FP fusions by anti-GFP.
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To confirm the expression of desired products, recombinant proteins were probed using anti-GFP via standard western blot techniques. Protein concentrations were normalized to an established loading control, β-actin, which provides a means of direct comparison among various fusion proteins (Figure 2B). Notably, the parental strain of yeast had a profound effect on the concentration (i.e. molecules/cell) of BiP-FP when observed at an identical stage of growth. This difference was most likely a result of the degree to which ERQC mechanisms were initiated in various parental strains. Protein levels of BiP are increased during cell stress as a consequence of transcriptional upregulation following UPR or HSR activation. We established that the UPR is initiated earlier in BJ5464 cells – the strain lacking vacuolar proteases essential for the degradation of aberrant proteins – as compared to BY4742 by implementing classical UPR sensors [4, 78]. A greater magnitude of the response (i.e. BiP protein levels and fluorescence of the UPRE sensor) was observed in this protease-deficient strain (data not shown). Data in Figure 2 are consistent with these results.
Previously, the oxidative environment of microbial and eukaryotic cells has resulted in either a lack of fluorescence , oligomerization  or mixed disulfides  for FP fusions. We evaluated all fluorescent cassettes within the ER by in-gel and western blot analyses (Figure 2C,D) under non-reducing conditions to examine the possibility of oligomerization or disulfide formation. As shown, all strains that recombinantly expressed BiP-FP fusions displayed one product regardless of whether the probe of interest was a GFP variant, a designed monomer (e.g. yEmCFP or yEmCitrine that contains the A206R substitution to prevent dimerization), or mCherry (DsRed variant) that does not contain cysteines exposed to the lumen.
Growth rates were calculated for BiP fused to Cerulean, Venus or mCherry as an indirect measure of whether FP-fusions adversely affected cell physiology. Since previous studies by Townsley et al.  demonstrated that modification of the ER retrieval sequence compromised strain viability, a characteristic generation time of each FP fusion was compared to S. cerevisiae strain BJ5464 [i.e. wildtype (WT)]. In Figure 3A, the mean optical density (OD600) is shown for each time point sampled and error bars depict the minimum and maximum measurement from three experimental replicates. The semi-log plot of Figure 3A shows that the growth rate of BiP-Cerulean and BiP-mCherry is slower than either BiP-Venus or the parental strain. Two-sample t-tests statistically confirmed this hypothesis. As shown in Figure 3A (inset), vertical bars represent the mean doubling time for each strain whereas the corresponding p-value is located above each bar. Error bars are defined as the 95% confidence interval of the mean. At the 5% significance level (α = 0.05), the mean doubling time of BiP-Venus (99.7 min) was equal to the WT strain, BJ5464 (102 min). In contrast, the doubling times of BiP-Cerulean (133 min) and BiP-mCherry (122 min) were greater than the control. Interestingly, when time course experiments were performed for BiP fused to yeast-enhanced variants (e.g. yEmCFP, yEmCitrine and yEGFP implementing the codon-optimized polylinker), doubling times of both monomeric variants were comparable to the WT strain. Two-sample t-tests confirmed the equality of mean doubling times for WT, yEmCFP and yEmCitrine strains (Figure 3B, inset). Although these results indicate minor perturbations to the system, increased doubling times do infer some impact on the cellular physiology when BiP was fused to Cerulean, mCherry or yEGFP.
Figure 3. Evaluation of growth rates for Saccharomyces cerevisiae strains constitutively expressing BiP/Kar2p fluorescent protein fusions. A) Time course experiments were performed for parental strain BJ5464 (filled circles, WT) and derivatives consisting of BiP/Kar2p fused to Cerulean (filled squares, CY2007), Venus (open triangles, CY2009) and mCherry (filled diamonds, CY2014). Experiments were completed in triplicate and graphically represented as a semi-log plot. For each time point, the mean ± error (i.e. minimum or maximum value of the measured OD600) is represented. To statistically evaluate the respective doubling times, two-sample t-tests were performed at the significance level of α = 0.5. Results are shown in the inset of (A), where the mean doubling times, , are denoted by the vertical bars with error bars defined as the standard error of the mean. Statistical significance results in the corresponding p-values (numbers above vertical bars). B) Time course experiments were performed for parental strain BJ5464 (filled circles, WT) and derivatives consisting of BiP/Kar2p fused to yeast-enhanced variants yEmCFP (filled squares, CY2016), yEmCitrine (open triangles, CY2012), and yEGFP (filled triangles, CY2013). Similar to (A), experiments were completed as replicates and doubling times were calculated following 18-h post-inoculation, as described in Materials and Methods. Two-sample t-tests were also performed for yeast-enhanced variants fused to BiP/Kar2p, and results are shown in the inset of (B). C) Immunofluorescence depicts the localization of endogenous BiP in the ER. The ER localization of BiP was probed with anti-Kar2p in all three parental strains used in this study, detailing three subcompartments: nuclear ER, peripheral ER, and connecting tubules. D) Live-cell imaging of BiP FPs require the appropriate ER retrieval sequence (i.e. FEHDEL). Fluorescence (left) and DIC (right) images of the indicated strains acquired by confocal microscopy (Zeiss 780 confocal microscope, 100×/NA 1.46), as described in Materials and Methods. All scale bars are 5 µm.
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The ER localization of endogenous BiP was determined originally by immunofluorescence techniques using anti-Kar2p primary antibodies, as published by Rose and colleagues . To correlate the intracellular localization of BiP fused to FP variants in several S. cerevisiae strains, we first probed for the endogenous species using anti-Kar2p . As depicted in Figure 3C, immunofluorescence of all parental strains defined three ER subcompartments (i.e. perinuclear ER, cortical ER and tubular connections) when imaged by confocal microscopy. Furthermore, live-cell imaging of FP variants did not cause mislocalization. By comparison to the immunofluorescent images, it is apparent that all BiP-FP fusions were correctly localized to the ER, as demonstrated in Figure 3D. In contrast to immunofluorescent techniques, the implementation of BiP-FP variants with retrieval motifs provides a distinct advantage as it facilitates time-lapse imaging, enabling real-time protein monitoring during cell homeostasis and immediately following disturbances to the system (e.g. activation of ER stress). Live-cell images of BiP-FPs assessed during early- to mid-log phase depicts a spatial distribution vastly similar to immunofluorescence. However, under conditions of nutrient deprivation during stationary phase, (>1.8 OD) BiP distributes to form larger inhomogeneities within the ER, a phenomenon that has been assessed previously for UPR complexes [82, 83]. FRAP experiments (Video S1) showed that intracellular BiP clusters are localized to the ER based upon the recovery of fluorescence within the region of interest (ROI) photobleached.
Within the early secretory pathway of S. cerevisiae, Pelham and colleagues  established that the HDEL recognition system can become saturated. Previous studies confirmed that endogenous BiP is secreted during overexpression of an HDEL-tagged prepro-alpha factor. To ensure that the retrieval motif of our cassette series is recognized and that retrograde transport machinery is not overwhelmed, time course experiments were performed for all BiP-FP variants. When cultures attained log-phase growth, the media was probed for BiP by standard western blot techniques. Our results indicated that BiP was not secreted in the WT strain. Interestingly, for BiP-FP fusions expressed under the regulation of the native KAR2 promoter, no detectable secretion was observed (data not shown). Collectively, these results confirm that the HDEL receptor effectively recognized modules containing the retrieval motif.
The utility of our cassettes was further validated when applied to the ER foldase, Pdi1p. All FP variants containing C-termini HDEL retrieval sequences were directed to the PDI1 ORF. Similarly, methodologies described above were completed for Pdi1p. In Figure 4A, each Pdi1p-FP fusion was distinguishable via an in-gel fluorescent technique. Western blot analysis verified the expression of fusion proteins derived from GFP (Figure 4B). In order to normalize each species for direct comparison, a subsequent analysis probed for the Tubulin loading control. Of particular note is the decreased fluorescence intensity of blue variants (Figure 4A) compared to the results of the GFP primary antibody and established loading control (Figure 4B). Similarly in Figure 4A, the fluorescence intensity of Pdi1p-mCherry-HDEL expressed in BY4742 was significantly less than its intensity in BJ5464, which is a direct result of total protein concentration probed by anti-Tubulin. The decreased recovery of BY4742 Pdi1p-mCherry-HDEL may be partially due to different growth rates of these parental strains. In contrast to our investigations of BiP-FP – specifically the lack of degraded FP variant, mixed disulfides or oligomerization – Pdi1p-Venus does contain a fluorescent degradation product. Effectively, 36% of total recombinant Pdi1p expressed is degraded and detected via in-gel fluorescence and western blot analysis (data not shown).
Figure 4. Expression of Pdi1p fluorescent protein fusions that incorporate the C-terminal HDEL retrieval sequence. A) In-gel fluorescence analysis of GFP and DsRed variants fused to protein disulfide isomerase, Pdi1p, compared to parental strains. FP fusions were detected at excitation wavelengths of 457, 488, or 532 nm. B) Immunoblots detect fluorescent protein fusions. Western blot analysis shows corresponding lanes analogous to (A); protein bands were probed with anti-GFP and compared to total protein via an appropriate loading control, anti-Tubulin. C) Live-cell imaging of Pdi1p fluorescent protein fusions localized to the ER. Fluorescence (left) and DIC (right) images of the indicated strains acquired by confocal microscopy, as described in Materials and Methods (Zeiss 780 confocal microscope, 100×/NA 1.46). D) Implementation and optimization of blue fluorescent protein variants, yECFP and yEmCFP, fused to Pdi1p. Live-cell imaging of Pdi1p fusion proteins – fluorescence (left) and DIC (right) – were acquired with improved sensitivity provided by GaAsP detectors (Zeiss 780 confocal microscope, 100×/NA 1.46) at an excitation of 458 nm. All scale bars are 5 µm.
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Although live-cell imaging adequately detected FP variants yEGFP, Venus, yECitrine and mCherry during the live-cell imaging of recombinant Pdi1p (Figure 4C), additional fluorescent aspects are noteworthy. For example, the intensity of blue GFP variants yECFP and yEmCFP was not detected prominently via confocal microscopy (Figure 4D) under the parameter settings described in Materials and Methods. These results are in agreement with the decreased fluorescence intensity of blue variants, as examined by in-gel fluorescent techniques and confirmed by western blot procedures (Figure 4A,B). We have previously demonstrated the significance of the increased sensitivity provided by gallium arsenide-phosphide (GaAsP) detectors , which are twofold more sensitive than standard photomultiplier tubes (PMTs) . Thus, the use of Pdi1p fused to either yECFP or yEmCFP reveals a dramatic limitation for experiments involving time-lapse imaging. The degradation of GFP variants was observed in the vacuole of green, yellow and blue FPs fused to Pdi1p (Figure 4C); however, DsRed variants are not detected appreciably within the vacuole when expressed as a Pdi1p-FP fusion.
KDEL retrieval is used for ER co-chaperone, Scj1
Interestingly, the mammalian homolog of BiP (i.e. Grp78) contains a C-terminal KDEL retrieval sequence even though the receptor actually binds the tetrapeptide HDEL more tightly [86, 87]. Pelham and colleagues  determined varying affinities of retrieval sequences (i.e. FEHDEL, SEKDEL, SAEARRL, etc.) for retrograde transport machinery. Hence, there exists some variation in the first two positions of the motif, which has been proposed to affect the rate of retrieval amongst resident proteins  (Table 1). In S. cerevisiae, only Scj1p – an ER luminal co-chaperone involved in protein folding and maturation – contains this KDEL motif. To determine the impact of trafficking sequences, we implemented a C-terminal MLKDEL sequence and investigated Scj1 FP fusions. In-gel fluorescence distinguished between parental strains and the recombinantly expressed Scj1p FP variants (Figure 5A). Our analyses with Scj1p indicated that in-gel fluorescent methods are an adequate means of assessing protein levels that are significantly less than mm concentrations in the luminal environment. In Figure 5B, western blot analysis confirmed the expression of GFP variants. To normalize each species for direct comparisons, subsequent analysis probed for the loading control, β-Actin. To our knowledge, Scj1p has not been analyzed in vivo; thus, live-cell imaging techniques of Figure 5C demonstrate the successful trafficking of Scj1p within the early secretory pathway of yeast. We have confirmed these results via examination of three common strains. Images (Figure 5C) suggest that Scj1-FP does traffic to the vacuole whereas in-gel fluorescent analyses detect a low percentage (i.e. 28%) of degraded FP variant, as compared to total expression of Scj1p-FP. Of particular note during live-cell imaging, we utilized GaAsP detectors in order to visualize endogenous levels of Scj1p FP fusions. This experimental condition may limit the utility of this construct for proposed experiments involving time-lapse microscopy.
Figure 5. Expression of Scj1p GFP variants that implement the C-terminal KDEL retrieval sequence. A) In-gel fluorescence analysis of yellow GFP variants fused to Scj1p compared to parental strains. GFP variants were excited at 488 nm. B) Immunoblot analysis of Scj1p fusion proteins. Western blot analysis shows corresponding lanes; protein bands were probed with anti-GFP and compared to total protein load via an appropriate control, anti-Actin. C) Live-cell imaging of Scj1p GFP variants. Fluorescence (left) and DIC (right) images of the indicated strains acquired by confocal microscopy (Zeiss 780 confocal microscope, 100×/NA 1.46), as described in Materials and Methods. All scale bars are 5 µm. D) Growth rates of Saccharomyces cerevisiae strains constitutively expressing ER-resident proteins fused to Venus with selective H/KDEL retrieval sequences. Time course experiments were performed for parental strain BJ5464 (filled circles, WT) and derivatives consisting of BiP/Kar2p (open triangles, CY2009), Pdi1p (open squares, CY2017), and Scj1p (open diamonds, CY2021) fused to Venus (e.g. pCY3051-01 or pCY3052-01, respectively). Experiments were completed in triplicate and graphically represented as a semi-log plot. For each time point, the mean ± error (i.e. minimum or maximum value of the measured OD600) is represented. To statistically evaluate the respective doubling times, two-sample t-tests were performed at the significance level of α = 0.5. Results are shown in the inset of (D), where the mean doubling times, , are denoted by the vertical bars with error bars defined as the standard error of the mean. Statistical significance results in the corresponding p-values (numbers above vertical bars).
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Strep-tag II purification of an ER chaperone
In addition to FP variants and epitope tags, small affinity peptides enable the rapid isolation of recombinant gene products when coupled with effective procedures for detection, purification and immobilization . Strep-tag II is a versatile affinity peptide that does not impede protein translocation nor inhibits protein-folding events, and is largely resistant to cellular proteases . As compared to the original Strep-tag [i.e. WRHPQFGG, ], Strep-tag II permits greater flexibility in its choice of attachment site  and is used with an engineered variant of streptavidin, StrepTactin, which has an improved peptide binding capacity . These attributes strongly advocate the implementation of Strep-tag II to detect and purify ER folding factors.
To characterize the biochemical activity of BiP and Pdi1p, existing purification strategies employ either multistep purification methods [i.e. utilizing the affinity of a BiP/Kar2p specific antibody combined with hydroxyapatite column chromatography  and isolation of endogenous Pdi1p in S. cerevisiae via precipitation followed by affinity chromatography ] or strong denaturing conditions [e.g. N-terminal His6-tagged BiP expressed in E. coli and purified by Ni+2-NTA affinity chromatography ]. In contrast, we incorporated an eight-amino acid yeast-codon optimized peptide (WSHPQFEK) referred to as Strep-tag II , and utilized the StrepTactin affinity method.
Prior to this study, S. cerevisiae ER resident proteins BiP and Pdi1p were detected solely by standard techniques, using primary antibodies derived from the endogenous gene. It is often desirable to detect Strep-tag II fusions with high sensitivity – by either standard immunoblotting techniques, immunofluorescence or flow cytometry – for which conjugated StrepTactin/enzyme substrates are commercially available . Alternatively, monoclonal antibodies such as StrepMAB-Classic (IBA BioTAGnology) permit the sensitive detection of fusion proteins. In Figure 6A, StrepMAB-Classic was used to probe for the expression of BiP-Strep-tag II - HDEL, which was detected at the predicted molecular weight of 76.9 kDa. As illustrated in Figure 6B, the cell lysate, which consists of recombinantly expressed BiP and host proteins, was immobilized to StrepTactin. Non-specifically bound proteins were removed by sequential washes. The desired product was then eluted under mild conditions, via competition with D-desthiobiotin. In Figure 6C, the results of an efficient BiP-Strep-tag II purification strategy are shown. Approximately 80 OD600 (equivalent to 80 × 107 cells) yielded a 54% recovery of total BiP from the lysate (eluates 1–5), while less than 1% of product remained bound to the resin. Additionally, 34% of the product resided in the flow-through thereby providing a basis from which to optimize future studies.
Figure 6. Recombinant BiP was successfully purified from Saccharomyces cerevisiae under physiological conditions. A) Expression of BiP-Strep-tag II, designed with an HDEL retrieval motif. Western blot analysis of ER molecular chaperone BiP/Kar2p, expressed as a recombinant Strep-tag II fusion protein, compared to WT. Protein bands were probed with anti-Strep-tag® II. B) Illustration of Strep-tag II/StrepTactin purification, schematic modified from Schmidt and Skerra . Cell lysate containing host proteins and recombinant BiP is applied to immobilized StrepTactin. Host proteins are removed by sequential washes. BiP-Strep-tag II - HDEL is then displaced via competition with a low concentration of D-desthiobiotin. To regenerate the column, the removal of D-desthiobiotin is accelerated by the addition of HABA. C) Purification strategy and recovery of BiP-Strep-tag II - HDEL. Analysis of recombinantly expressed and purified BiP-Strep-tag II - HDEL. Initially, cleared cell lysate (Load, 4.6 mLs total) was applied to StrepTactin resin (2.5 mLs column volume) and flow-through (FT, 4.6 mLs) was collected. The column was washed sequentially five times (W, 5 mLs each), followed by the elution of BiP-Strep-tag II - HDEL (E, 8 x 1.25 mLs with 2.5 mm D-desthiobiotin). All fractions were normalized to volumes used throughout the purification scheme.
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Applications of pCY H/KDEL cassettes
The molecular mechanisms underlying ER homeostatic regulation have been identified, but how they coordinate the comprehensive restructuring of ER resident proteins via transcriptional regulation and remodeling of organelle morphology is only partially understood. To experimentally address these questions, we have designed and implemented a new series of versatile PCR-based modules for C-terminal tagging of ER resident proteins in yeast. Collectively, we established that in-gel fluorescence is an efficient method to evaluate recombinantly expressed ER resident proteins fused to FP variants, prior to confirmation by western blot analyses. Moreover, live-cell imaging techniques affirmed the correct localization of BiP, Pdi1p and Scj1p fusions proteins. The viability of each novel strain expressing recombinant fusions was quantified by time course experiments, from which doubling times were statistically evaluated. Neither Pdi1p fused to blue variants nor Scj1p fused to yellow variants possessed adequate fluorescence for time-lapse imaging; therefore, we have identified limitations associated with the choice of FP in the ER luminal environment. We successfully demonstrated that affinity purification was achieved for BiP-Strep-tag II - HDEL under physiological conditions. Combined, the pCY H/KDEL cassettes enable the measurement of protein concentrations compatible with western blot analyses, utilize optimal fluorescence spectra and selection for multicolor labeling in vivo, facilitate protein trafficking studies in the secretory pathway, and investigate select protein interactions.
We envision this series of modules to be an invaluable tool, broadly applicable, to yeast researchers investigating the role of ER resident proteins in cell homeostasis, ER-to-Golgi trafficking, protein structure/function relationships of purified ER resident proteins, and the kinetics of ER molecular chaperone/co-chaperone interactions. Interestingly, the implementation of H/KDEL modules could effectively sense the activation of cellular quality control mechanisms in vivo, similar to results achieved by existing UPR sensors fused to GFP or DsRed variants [4, 78] and recently demonstrated by Lajoie et al. . Prolonged UPR activation is linked to pathophysiological processes, as well as a number of prevalent diseases [reviewed in [96, 97]] highlighting UPR modulation as an emerging focus of therapeutic design . Notably, many components of neurodegenerative and hereditary diseases have been studied in S. cerevisiae [99-102], thus advocating the use of this cassette series in a model eukaryote to investigate cell physiology and disease.