Subcellular localization of selected NST like proteins

Nucleotide sugar transporters reside in the membrane of the Golgi apparatus and/or endoplasmic reticulum. Subcellular localization of our putative transporters can thus be used as an additional selection step as well as part of initial characterization of these proteins.

3.1.4.1 Expression of predicted NSTs in CHO K1 cells

To explore the cellular segregation of the selected nine proteins, the cDNAs encoding the complete open reading frames cloned in the pcDNA3 vectors were transfected in CHO K1 cells.

Resulting plasmids lead to expression of N-terminally FLAG tagged proteins under regulation of the strong CMV promoter. Transient expression of the proteins and immunostaining with the anti-Flag antibody in CHO K1 cells were performed as described in material and methods. Two splice variants of the human UDP-Gal transporter called UGT1 and UGT2 were used as control.

The former resides in the Golgi apparatus, while the second localizes both in the ER and Golgi.

Additionally the Golgi resident alpha-Mannosidase II was used as Golgi marker. As shown in figure 10, hORF-11 (SLC35B4) co-localizes with this protein and is thus localized in the Golgi apparatus like the UGT1 control whereas the distribution of hORF-15 (SLC35C2) is similar to UGT2 indicating both endoplasmic reticulum and Golgi apparatus localization.

Results

Figure 10: Subcellular localization of SLC35B4 and SLC35C2. CHO cells were transiently transfected with FLAG-tagged UGT1, UGT2, SLC35B4 and SLC35C2. The localisation of the recombinant proteins was analysed by indirect immunofluorescence microscopy. The FLAG tagged SLC35B4 transporter (red) co-localises with the Golgi marker mannosidase II (green). The SLC35C2 protein shows a distribution similar to UGT2 (control) and therefore is suggested to be localised in the ER and Golgi.

Over-expression of other hORFs seems to destroy the internal organization of the cells leading to cell death as the only stained cells were small rounded cells without apparent Golgi apparatus. In order to decrease protein expression, experiments were first repeated with a lower

concentration of plasmid DNA and shorter time of expression but the results did not improve.

We then tried to generate “semi stable” cell lines to limit the plasmid copy numbers and lower protein expression. The remaining hORFs were therefore transfected in CHO K1 cells and antibiotic selection was applied. Only cells with low expression of the transporter are expected to survive. Unexpectedly, two days after transfection no cells were alive. Results obtained from this experiment show clearly that expression of putative transporters under control of CMV promoter is too high and kills the cells. A system in which the level of hORFs expression could be tightly controlled is thus required for localization of NSTs in mammalian cells.

3.1.4.2 Expression under control of the tet repressor

The T-REx™ System is a tetracycline-regulated mammalian expression system that uses regulatory elements from the E. coli tetracyclin resistance operon (Hillen et al., 1983; Hillen and Berens, 1994).The regulation is based on the binding of tetracycline repressor homodimer to the tetracycline operator inserted after the strong CMV promoter as is indicated on figure 11. The ratio tetracycline – repressor determine the level of expression of the gene of interest (in our case NSTs). The repressor protein is encoded from pcDNA6/TR vector and is under regulation of the CMV promoter. By applying different concentration of tetracycline, the amount of produced NST can vary from virtually zero to the level obtained from the CMV promoter.

The cDNA of nine putative transporters listed in table 1 were recloned in pcDNA4 vector (utilizing the same multiple cloning sites) in witch the CMV promoter is repressed by Tet – operator. Human ORFs and pcDNA6 were co-transfected in CHO cells and antibiotic selection was applied. Using this approach hORF-7 (SLC35E3) was successfully localized in the Golgi apparatus (figure 12).

Results

Figure 12: Subcellular localization of SLC35F6. CHO cells were transiently cotransfected with FLAG-tagged SLC35F6 and pcDNA6/TR and antibiotic selection was applied. The localisation of the recombinant protein was analysed by indirect immunofluorescence microscopy. FLAG tagged SLC35F6 (red) co-localises with the Golgi marker mannosidase II (green).

Figure 11: Inducible expression of the gene of interest under regulation of the tet repressor. The tet repressor (tetR) is expressed from pcDNA6/TR in transfected cells. TetR homodimers bind to tet operator (tetO2) sequences and the expression of the gene of interest is suppressed. Tetracycline, added in the medium binds to tetR and induces conformational changes in the repressor protein which results in releasing of the tetO2 sites. This leads to expression of the gene of interest.

However, the other proteins remain with unknown cellular distribution. Therefore, a system with the Tet-repressor stably integrated in the genome was used. For this, HeLa cells were used, as these are better suitable for intracellular localization of proteins. To generate the stable cell line, HeLa cells were transfected with pcDNA6/TR and selected with 0.7 mg/ml Zeocin antibiotic for stable integrants. Two days after transfection the cells were diluted and cultured till formation of single-cell colonies. Expression in selected single cell colonies was tested by transient transfection with pcDNA4/TO/LacZ. Cells were stained with X-gal 2 days after transfection. Forty-eight colonies were studied for expression / repression of LacZ. Two clones show high staining with x-gal in the presence of tetracycline and full repression without tetracycline. One of these clones (HeLa-TRex1) was used for further localization of putative transporters. Human ORFs in pcDNA4/TO were transfected as previously described. Different concentrations of tetracycline were applied. As expected addition of 1 mg/ml of the tetracycline induces maximal protein expression and kills transfected cells. Surprisingly, the same effect was observed with 0.5 and 0.05 mg/ml concentrations. However, small numbers of cells transfected with SLC35B1 were detected in the induction experiment with 0.05 mg/ml. Results shown in figure 13 suggest Golgi localization of SLC35B1. Expression without tetracycline was observed in some cells but the amount of protein was at the limit of the detection system. The other candidate NSTs could not be successfully localized.

Figure 13: Subcellular localization of SLC35B1. HeLa cells stably expressing tet repressor were transiently transfected with FLAG-tagged SLC35F6. To induce the expression, 0.05 mg/ml of tetracycline was applied.

Localisation of the recombinant protein was analysed by indirect immunofluorescence microscopy. The FLAG tagged SLC35B1 transporter (red) co-localises with the Golgi marker mannosidase II (green).

Results