hPar14 is localized in the cytoplasm and the nucleus

Since hPar14 was discovered, there is no direct information about the subcellular localization of this protein in cells and its putative function. To gather information about the intracellular localization of endogenous hPar14, HeLa cells were fractionated according to the protocol described in Materials and Methods. The total cellular protein concentration was estimated using Coomassie Protein Assay. Equal amount of cytoplasmic and nuclear fractions were subjected to 15 % SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-hPar14 rabbit serum, anti-α tubulin (control for cytoplasmic preparation) and anti-c-myc (control for nuclear preparation) (Figure 3.1).

Figure 3.1 Localization of hPar14 in HeLa cells detected by Western blot. HeLa cells were fractionated and equal amount of cytosolic and nuclear fractions were separated by SDS-PAGE and analysed by Western blot using anti- c-myc (1:100), anti-α-tubulin (1:500) and anti-hPar14 rabbit serum (1:1000) antibodies.

The fractionation of the HeLa cells revealed that hPar14 is present in both cellular fractions:

cytosolic and nuclear, with higher abundance in the nucleus as detected by Western blot analysis. This is in agreement with previous observations where a relatively high level of endogenous hPar14 was demonstrated in the nucleus compared to cytoplasm using electron microscopy (Thorpe, et al., 1999). To estimate the difference in amount of hPar14 between cytosolic and nuclear fractions, a densitometry analysis of the Western blot data was

64 kDa

50 kDa

14 kDa c-myc

α-tubulin

hPar14

CYTOSOL NUCLEAR

MW

performed (Figure 3.1), using Advanced Image Data Analyser AIDA 2.0. This analysis revealed that the amount of hPar14 is two-fold higher in the nucleus than in the cytoplasm (Figure 3.2).

Figure 3.2 Relative amount of hPar14 in cytosolic and nuclear fraction obtained from HeLa cells. Black line indicates mean error.

To test whether the in vivo distribution of hPar14 is similar to that observed in vitro, a DNA construct encoding hPar14 fused on its carboxy terminus to the green fluorescent protein (GFP) of the jellyfish Aequorea victoria was developed and expressed in HeLa cells. Figure 3.3 shows fluorescence image of HeLa cells transfected with the hPar14-GFP construct and analysed by fluorescence microscopy. The most intensive green light emission comes from the nucleus but the protein is also detected in the cytoplasm.

Figure 3.3 Fluorescence image of HeLa cells transfected with hPar14-GFP. The right panel, hPar14-GFP expressed in HeLa cells. Cells transfected with DNA construct encoding hPar14-hPar14-GFP, were fixed

PPIase domain

N-terminal extension

GFP 1-35 aa 36-131aa

hPar14-GFP

hPar14-GFP

and analysed by fluorescence microscopy. GFP images were collected using confocal fluorescence microscope at 60x water immersion C Apochromat objective. The picture shows green fluorescence of transfected cells. Three independent experiments showed the same results. The left panel, scheme of hPar14 fused to GFP.

To eliminate potential artefacts that might result from expression of the recombinant protein, we examined the endogenous hPar14 protein using affinity purified anti-hPar14 serum. The immunofluorescence analysis confirmed nuclear localization of endogenous hPar14 (Figure 3.4) but low cytoplasmic staining of hPar14 is also detected. As an additional control, the localization of hPin1 that is known to be an exclusive nuclear protein was tested. The localization pattern of hPin1 is very similar to that of hPar14. The protein is mainly detected in the nucleus. Some cytoplasmic staining is also observed.

Endogenous hPar14

Endogenous hPin1

Figure 3.4 Immunofluorescence localization of endogenous hPar14 and endogenous hPin1. HeLa cells were fixed, blocked, permeabilized and stained overnight with affinity purified anti-hPar14 or anti-hPin1

PI (DNA) hPar14-FITC Merge

PI (DNA) hPin1-FITC Merge

serum. Followed staining with anti-rabbit secondary antibody coupled to FITC and propidium iodide (PI) in the presence of RNAse, cells were analysed using confocal fluorescence microscopy with objective C-Apochromat 63x/1.2 W corr.

3.1.1 The 14 amino acids of hPar14 N-terminal are necessary for nuclear localization Shuttling of proteins between the cytoplasm and the nucleus can be achieved in two different ways. Relatively small proteins of molecular weight 14 kDa, e.g. parvulin, may be able to diffuse freely into the nucleus on a minute time scale (for review see Gama-Carvalho &

Carmo-Fonseca, 2001). The influx occurs through the nuclear pore complexes, which are supramolecular assemblies, embedded in the nuclear envelope. Selective entry into the nucleus would require a specific nuclear localisation signal (NLS), which induces gating of the pore complex and rapid passage into the compartment. Such signals are either short basic stretches of up to eight amino acids or bipartite sequences consisting of two basic segments separated by about ten less-conserved amino acids. Many of these targeting sequences are found in the C- or N-termini of their corresponding molecules.

The unstructured N-terminal domain of hPar14 exhibits a basic stretch of residues. Rulten and co-workers suggested that the extension might target the protein to the nucleus (Rulten, et. al 1999). Therefore, a study on the influence of N-terminal truncation of hPar14 on its cellular localization was conducted. Figure 3.5 A shows fluorescence images of HeLa cells transfected with hPar14-GFP constructs, wherein the first six ∆N6hPar14(6-131)-GFP and the first fourteen

∆N14hPar14(14-131)-GFP N-terminal residues were deleted. ∆N6hPar14(6-131)-GFP which lacks the first six amino acids is equally distributed through the nuclear matrix. The intracellular localization of ∆N14hPar14(14-131)-GFP is different from those observed for WT-protein and

∆N6hPar14(6-131)-GFP. The fluorescence image of ∆N14hPar14(14-131)-GFP shows a cytoplasmic localization of the recombinant protein. The molecules seem to accumulate in the nuclear envelope and the entry to the matrix is disturbed (Figure 3.5 B). In addition, we tested the expression of full-length and truncated forms of hPar14 fused to GFP by using Western blot with anti-GFP antibody. As is shown in Figure 3.5 B, all indicated proteins are expressed and have the expected molecular mass.

∆N6hPar14_(6-131)-GFP

6-35aa 36-131aa

A)

PPIase domain N-terminal extension

GFP Nucleus

Cytoplasm N-terminal extension PPIase domain

Localiaztion

Figure 3.5 The subcellular localization of truncated hPar14-GFP in HeLa cells. A) Schematic representation of the ∆N6hPar14(6-131)-GFP and ∆N14hPar14(14-131) -GFP. Middle panel, the fluorescence image of HeLa cells transfected with hPar14-GFP constructs, fixed and analysed by confocal fluorescence microscopy at C-Apochromat 63x/1.2 W corr objective. Three independent experiments showed identical results. B) Expression of hPar14 and its truncated form fused to GFP analysed by Western blot.

Thus, the signal for proper nuclear distribution of hPar14 is restricted to amino acids Ser7 to Lys14, which does not contain a classical NLS sequence. Despite the fact, that residues Ser7 to Lys14are responsible for the entry to the nucleus, hPar14 would be able to diffuse freely from the nucleus back to the cytoplasm. Retention of the protein could be due to binding to large, non-diffusible elements, e.g. the chromatin structure, which would explain the higher content of hPar14 in the nuclear fraction when compared to the cytoplasmic fraction.

∆N6hPar14(6-131)-GFP ∆N14hPar14(14-131)-GFP

B)