KNOCK ‐ OUT IN CELL LINES VIA TALEN TECHNOLOGY
III.1. C HARACTERIZATION OF THE HUMAN A LDO ‐ K ETO
III.1.3. S UBCELLULAR LOCALIZATION OF AKR1B15 ISOFORMS
III.1.3. S
UBCELLULAR LOCALIZATION OFAKR1B15
ISOFORMSMost members of the AKR superfamily, like AKR1B10 (sharing 91 % amino acid sequence identity with AKR1B15.1), localize intracellular to the cytosol. In order to fathom the subcellular localization of the two AKR1B15 isoforms, immunocytochemical analyses and in silico predictions were carried out.
SU B CE L L U L A R L O C A L I Z A T I O N O F F U L L L E N G T H AKR1B15 IS O F O R M S Subcellular localization studies with HeLa cells overexpressing N‐ and C‐terminally myc‐tagged AKR1B15 isoforms showed that both N‐terminally myc‐tagged AKR1B15 isoforms, AKR1B15.1 and AKR1B15.2, colocalize with the cytosol [Figure III‐9A, B]. A cytosolic localization was also determined for the C‐terminally myc‐tagged AKR1B15.2 [Figure III‐9D, F, H]. In contrast, AKR1B15.1 carrying a C‐terminal myc tag colocalized surprisingly with mitochondria [Figure III‐9C, E, G].
Since it was found that the location of the fused myc tag within the protein influenced the subcellular localization of AKR1B15.1 – cytosolic versus mitochondrial – localization studies were also performed with untagged AKR1B15 isoforms. These studies dealt with two questions: First, what are the subcellular localizations of untagged AKR1B15 isoforms and second, are the generated monoclonal rat‐anti‐AKR1B15 (AKB‐2) antibodies also functional in immunocytochemical analyses. The subcellular localization studies with untagged proteins revealed that, comparable to the C‐terminally myc‐tagged AKR1B15 isoforms, untagged AKR1B15.1 colocalizes to mitochondria [Figure III‐10A, C, E], whereas untagged AKR1B15.2 is a cytosolic protein [Figure III‐10B, D, F]. Thus, the mitochondrial localization of AKR1B15.1 was verified.
Figure III‐9: Immunocytochemical subcellular localization studies with myc‐tagged AKR1B15 isoforms revealed a surprising mitochondrial localization of C‐terminally myc‐tagged AKR1B15.1.
Shown are representative results from subcellular localization studies with N‐ or C‐terminally myc‐tagged AKR1B15 isoforms. HeLa cells were transiently transfected with N‐myc‐pcDNA3‐AKR1B15.1 (A), N‐myc‐
pcDNA3‐AKR1B15.2 (B), pcDNA4‐myc/His B‐AKR1B15.1 (C, E, G), or pcDNA4‐myc/His B‐AKR1B15.2 (D, F, H) in order to express N‐terminally myc‐tagged (A‐B) or C‐terminally myc‐tagged proteins (C‐H).
Nuclei were stained using Hoechst 33342 dye (a); counterstaining of the cytosol or endoplasmic reticulum was performed via the co‐transfection of pCMV DsRed‐Express2 or pDsRed2‐ER, respectively, whereas mitochondria were counterstained by vital stain using MitoTracker Orange CMTMRos (b); myc‐tagged AKR1B15 isoforms were stained using mouse‐anti‐myc / AlexaFluor 488 goat‐anti‐mouse antibodies (c).
The overlay of individual stains (d) shows nuclei in blue, counterstains in red, AKR1B15 in green, and colocalization in yellow.
Figure III‐10: Immunocytochemical subcellular localization studies with untagged AKR1B15 isoforms verified the mitochondrial and cytosolic localization of AKR1B15.1 and AKR1B15.2, respectively.
Shown are representative results from subcellular localization studies with untagged AKR1B15 isoforms.
HeLa cells were transiently transfected with either pcDNA3.1(+)‐AKR1B15.1 (A, C, E) or pcDNA3.1(+)‐
AKR1B15.2 (B, D, F) in order to express untagged AKR1B15 isoforms. Non‐transfected HeLa cells served as control (G). Nuclei were stained using Hoechst 33342 dye (a); mitochondria were stained using MitoTracker Orange CMTMRos (b); untagged AKR1B15 isoforms were stained using either the monoclonal rat‐anti‐
AKR1B15 clone 9A5 (A, B) or clone 19E5 (C, D) primary antibody supernatants and AlexaFluor 488 goat‐
anti‐rat secondary antibody pairs or the polyclonal rabbit‐anti‐AKR1B15 (E‐G) primary antibody and AlexaFluor 488 goat‐anti‐rabbit secondary antibody pair (c). The overlay of individual stains (d) shows nuclei in blue, mitochondria in red, AKR1B15 in green, and colocalization in yellow.
In addition, it could be seen that the generated monoclonal rat‐anti‐AKR1B15 clone 9A5 and clone 19E5 antibodies also work in immunocytochemical analyses by possessing sufficient affinity and low background [Figure III‐10A‐D]. Since the monoclonal antibodies were not existent at the beginning of these studies, the affinity purified polyclonal rabbit‐anti‐
AKR1B15 antibody was also included in these analyses. Although this antibody recognized both AKR1B15 isoforms in the subcellular localization studies when overexpressed in HeLa cells [Figure III‐10E, F], it gave also quite strong background signals resembling structures of the cytoskeleton [Figure III‐10G]. These observations were in accord with the results gained from Western blotting experiments [III.1.2.4].
In conclusion, whereas most AKRs, including the longer AKR1B15 isoform AKR1B15.2, are cytosolic proteins, AKR1B15.1 seems to be the first (human) AKR1 family member localizing to mitochondria.
IN S I L I C O S U B CE L L U L A R L O C A L I Z A T I O N P R E D I C T I O N
Immunocytochemistry revealed an unexpected mitochondrial localization of AKR1B15.1.
Although it is known that in silico subcellular localization predictions are error prone, different subcellular prediction algorithms (TargetP 1.1 Server, SignalP 4.1 Server, PrediSi, iPSORT, PSORT II, and MitoProt II) were used for the prediction of signal peptides or signal cleavage sites in AKR1B15.1 and the subcellular localization of AKR1B15.1 in order to underline the results gained from immunocytochemistry and to get an idea of key amino acid residues in AKR1B15.1 responsible for its mitochondrial localization. For comparison reasons, sequences of AKR1B15.2 and AKR1B10 were also included in the in silico analyses.
The TargetP 1.1 Server, SignalP 4.1 Server, iPSORT, and MitoProt II prediction algorithms did not predict any signal peptides or signal cleavage sites in the three proteins. However, a cleavage site with a score of 0.185 at position 26 of AKR1B15.1 was predicted by PrediSi.
Although that score was quite low and arguable, it was significantly higher compared with the scores of cleavage sites predicted for AKR1B15.2 (score of 0.074 at position 41) and AKR1B10 (score of 0.056 at position 221). When looking at the subcellular localization predictions, the TargetP 1.1 Server predicted no mitochondrial localization or secretory signal in AKR1B15.1, AKR1B15.2, and AKR1B10. The same was true for the PSORT II prediction algorithm which predicted a cytosolic localization for all three proteins.
MitoProt II predicted a mitochondrial localization of AKR1B15.1, AKR1B15.2, and AKR1B10 with a probability of 15.5 %, 0.8 %, and 12.0 %, respectively, very slightly promoting the results from the immunocytochemical analyses. In contrast, the iPSORT algorithm definitely predicted a mitochondrial targeting peptide in AKR1B15.1 but not in AKR1B15.2 and AKR1B10 by regarding the sequence of the first 30 amino acids. Thus, the iPSORT algorithm was the only prediction tool which clearly underlined the mitochondrial localization of AKR1B15.1, though AKR1B15.1 misses a distinct signal peptide. In addition, iPSORT indicated a crucial role of the N‐terminal amino acid residues in AKR1B15.1 for its subcellular localization. However, the in silico predictions represented per se just a poor tool to verify subcellular localizations.
The detailed outputs of predictions are listed in the appendix [VI.5].
EF F E CT OF N‐T E R M I N I O N T H E S U B C E L L U L A R L O C A L I Z A T I O N
The localization studies with myc‐tagged and untagged AKR1B15 isoforms as well as iPSORT predictions demonstrated that the N‐terminal amino acid sequence of AKR1B15.1 is responsible for its mitochondrial localization. To verify these results and identify amino acid residues important for the mitochondrial localization of AKR1B15.1 and the cytosolic localization of the highly identical AKR1B10, N‐terminal sequence stretches of AKR1B10, AKR1B15.1, and AKR1B15.2 were N‐terminally fused to AcGFP and these fusion proteins were analyzed for their subcellular localization by confocal microscopy [Figure III‐11, Figure III‐12].
The analyses showed that fusion proteins consisting of the first 38 amino acid residues (Met1‐Ala38) of AKR1B15.1 and AKR1B10 fused to AcGFP [Figure III‐11] colocalized with mitochondria and the cytosol, respectively [Figure III‐12A, D]. A cytosolic localization was also seen for the N‐terminus of AKR1B15.2 (Met1‐Ala66) fused to AcGFP [Figure III‐12E].
This reflected the earlier results from localization studies with full length proteins.
Figure III‐11: The N‐terminal amino acid sequences (Met1‐Ala38) of AKR1B15.1 and AKR1B10 differ in only two amino acid residues.
Shown are the alignment of the N‐terminal amino acid sequence (Met1‐Ala38) in AKR1B15.1 and in AKR1B10 as well as a schematic illustration of the N‐terminal sequence – AcGFP fusion proteins.
The two differing amino acid residues in the N‐terminal sequence of AKR1B15.1 and AKR1B10 at positions 22 and 24 are highlighted in purple.
As illustrated in Figure III‐4 and Figure III‐11, AKR1B15.1 and AKR1B10 differ in only two amino acid residues within the first 38 amino acid residues: Arg22 and Leu24 in AKR1B15.1 compared to Lys22 and Pro24 in AKR1B10.
To analyze which of the two differing amino acid residue is responsible for the cytosolic localization of AKR1B10 or, contrary, the mitochondrial localization of AKR1B15.1, Lys22 and Pro24 in the N‐terminal peptide (Met1‐Ala38) of AKR1B10 were separately mutated to the AKR1B15 equivalents Arg22 and Leu24, respectively. Here, it was seen that the K22R mutation in the sequence of AKR1B10 had no effect on the cytosolic localization of the fusion protein [Figure III‐12B]. In contrast, the P24L mutation changed the localization of the fusion protein towards mitochondrial [Figure III‐12C]. Thus, a single amino acid residue exchange, namely P24L, in AKR1B10 was sufficient to shift the localization of AKR1B10 from the cytosol to mitochondria.
Figure III‐12: Subcellular localization studies with N‐terminal sequence with AcGPF fusion proteins identified Leu24 in AKR1B15.1 versus Pro24 in AKR1B10 as key amino acid residues responsible for their mitochondrial versus cytosolic localization, respectively.
Shown are representative results from subcellular localization studies with N‐terminal AKR1B10 and AKR1B15 sequences fused to AcGFP. HeLa cells were transiently transfected with pAcGFP‐N1‐AKR1B10 (Met1‐Ala38) (A), pAcGFP‐N1‐AKR1B10 (Met1‐Ala38) K22R (B), pAcGFP‐N1‐AKR1B10 (Met1‐Ala38) P24L (C), pAcGFP‐N1‐AKR1B15.1 (Met1‐Ala38) (D), or pAcGFP‐N1‐AKR1B15.2 (Met1‐Ala66) (E) in order to express N‐terminal sequence – AcGFP fusion proteins. Nuclei were stained using Hoechst 33342 dye (a);
mitochondria were counterstained by vital stain using MitoTracker Orange CMTMRos (b); fusion proteins were visualized via AcGFP fluorescence (c). The overlay of individual stains (d) shows nuclei in blue, counterstains in red, AcGFP fusion proteins in green, and colocalization in yellow.
In conclusion, localization studies with the AKR1B15.1 N‐terminus (Met1‐Ala38) fused to AcGFP verified the mitochondrial subcellular localization of AKR1B15.1. In addition, Leu24 in AKR1B15.1 versus Pro24 in AKR1B10 was identified to be responsible for the mitochondrial and cytosolic localization of AKR1B15.1 and AKR1B10, respectively.