3.1. Post-translational membrane insertion of putative tail-anchored proteins
3.1.3. Investigation of INM-protein transport using an in vitro import assay
In addition to the biochemically performed membrane integration assays described in the above section 3.1.2, an in vitro transport assay was used to investigate membrane localization or at least association of bacterially produced TRC40/TA-protein complexes in a microscopy-based approach. The reaction performed in this experiment can be seen as the analogous or
“microscopic version” of the membrane insertion experiments in semi-permeabilized cells. This kind of in vitro transport assay used in this project, is a well-established method, which originally was used for studying either import or export of soluble proteins (Adam et al., 1990; Kehlenbach et al., 1998).
Besides the biochemical membrane insertion assays, the in vitro import assays were also performed in digitonin-permeabilized HeLa P4 cells (section 220.127.116.11) to examine, whether the
73 membrane integration, which was biochemically detected, could be visualized in cells as well.
The proteins were purified and co-expressed by the same means as described for the HZZ-emerin-opsin.
In the following, the membrane association of different emerin variants as well as VAPB and PTP1B were examined in these in vitro import assays using confocal microscopy.
Due to the specific co-expression and purification of the putative TA-protein of interest with TRC40, the proteins were in a kind of pre-determined state and were expected to also use the TRC40-pathway for membrane insertion. Therefore, a general localization of the studied proteins at least to the ER would be expected. Emerin, VAPB and PTP1B should not only be studied for being TA-proteins in terms of their membrane integration mechanism, but also in light of being INM-proteins and using the ER-membrane as a first entry site (Rapoport, 1992; Kutay et al., 1993) on their way to the INM. Furthermore, a localization not only to the ER but also to the nuclear envelope (seen by a “nuclear rim”) would consequently be expected for the selected proteins, given that “required” factors are available for targeting of a protein to the NE in this permeabilized cell-system.
Three different emerin versions were tested in this assay. Import reactions for mCherry-emerin-opsin, mCherry-FRB-emerin and a mCherry-Δ1-106-emerin-opsin each in a complex with MBP-TRC40 were prepared and the only two components added apart from the protein was an energy-regenerating system and cytosol (cytosolic extract from a HeLa suspension cell line). Each import reaction was performed directly on coverslips, on which HeLa cells were seeded one day prior to the assay. Generally, these import assays are performed at 37 °C and 4°C, because for all transport processes temperature can be a crucial parameter.
The mCherry-emerin-opsin was used, because the mCherry-tag provides an easy possibility to detect the protein at the microscope and this variant can be immunostained additionally for its C-terminal opsin-tag to determine localization inside the cell.
Furthermore, the MBP-TRC40/mCherry-FRB-emerin (section 18.104.22.168.1.1), was included, because of its possible usage in a rapamycin-INM-localization assay, which will be described in detail in chapter 22.214.171.124. Irrelevant of knowing the details and mechanism of this rapamycin assay, the localization of this variant compared to the first construct, which does not contain such a FRB-domain, were compared.
In addition to the full length (254 aa) versions of emerin, a shortened emerin variant with a deletion of the residues 1-106 was investigated, because it was reported to efficiently localize to the nuclear envelope even though, a large N-terminal portion of emerin was lacking (Tsuchiya et al., 1999).
When the import assays were performed at 37 °C and an energy-regenerating system as well as cytosolic extract was added, for all different emerin variants a localization at the ER and a NE could be observed (Figure 18). According to this, the soluble control cargo-protein BSA-NLS showed a temperature-dependent import, which was seen by an accumulation of Atto488-signal inside the nuclei and nucleoli. In contrast at 4 °C, the control protein showed no import into the nucleus and further for none of the emerin versions, a NE localization was detectable at this temperature. Instead a slight association of emerin to the ER seemed to occur and a
“punctate” pattern was observed. The strongest NE-localization was could be observed with the mCherry-Δ1-106-emerin-opsin deletion mutant (Tsuchiya et al., 1999). Comparing the
74 localization of mCherry-emerin with the mCherry-emerin-opsin variant lacking the FRB-cassette, no obvious difference in localization was seen, when looking at the anti-opsin compared to the anti-RFP staining (Figure 18, green signal). Altogether, for the analyzed emerin protein variants a localization or association at the ER membrane was detected and sometimes a more pronounced nuclear rim was observed indicating a localization at the nuclear envelope.
Other proteins, which were investigated for the possibility of transport to the nuclear membranes, were VAPB and PTP1B. Both were expressed as HZZ-tagged proteins. After performing the import reactions under the same conditions as for emerin, VAPB localized to a structure, which is most likely the ER (Figure 19, upper panel). Compared to emerin, where at least some part was observed at the NE, VAPB did not reveal any “nuclear rim” localization. A similar observation was made for HZZ-PTP1B-opsin, which showed no NE localization at all, although a perinuclear localization was detected (Figure 19, mid panel). The pattern observed for PTP1B was even more discontinuous than the “ER” localization observed for VAPB, therefore contrary to the observations made with in vitro translated PTP1B in membrane insertion assays, under these specific in vitro import assay conditions PTP1B might hypothetically only be associated and not integrated into the membrane.
Taken together, the membrane integration assays performed with in vitro translated proteins showed a possible post-translational insertion at the ER for emerin, LAP2β, VAPB and PTP1B.
Focusing on the membrane protein emerin, it was demonstrated that with either in vitro translated or bacterially expressed protein together in a complex with TRC40, a post-translational membrane insertion was utilized by emerin but not by VAPB in comparison. After having a closer look on the specific post-translational mechanism, the membrane integration of emerin was shown to depend on the TRC40-pathway, when inhibitory fragments of WRB and CAML were used in RM or SPCs independent of what kind of emerin variant (in vitro translated or purified in complex with TRC40) was used.
Additionally, depletion experiments showed, how strongly emerin depends on the TRC40 machinery. As a consequence, emerin is strongly suggested to belong to the group of tail-anchored proteins and seems to rely on the TRC40-mediated membrane integration.
Corresponding to the biochemical assays, an ER localization and thus as well a localization at the ONM which is continuous to the ER, is most likely to be seen for emerin in the in vitro import assay.
Overall, a statement about the exact subcellular localization of a protein is not possible on the basis of this method. These in vitro transport assays clearly illustrate the limitation of information provided concerning the exact subcellular localization of a protein.
As a consequence of this, the next chapter addresses the available options to determine the localization of a protein at the INM and presents a new approach for doing this.
76 Figure 18: In vitro transport assay to the NE using different emerin variants. Transport reactions of a TA-protein together with TRC40 to the ER membranes or NE were performed directly on the coverslips. One day prior to the transport assay, HeLa P4 cells were seeded on coverslips. After digitonin-permeabilization of the cells, the transport reaction was set up. The amount of protein in a complex with TRC40 used per import reaction varied for all different emerin fusion proteins (mCherry-emerin-op: 5 µg (1.8 µM);
mCherry-FRB-emerin: 1.8 µg (0.5 µM); mCherry-Δ1-106-emerin-op: 4.5 µg (2 µM)). The reaction was completed by addition of cytosol and ATP-regenerating system to the protein and the mixture was added to the cells. Transport was performed at 37 °C for one hour and an additional control reaction at 4 °C was prepared. Atto488-BSA-NLS served as a general control for the assay and should localize to the nucleoplasm as a soluble cargo protein. The fluorescent signal in the red (mCherry) or the green (α-opsin) channel indicates integration of the protein into the ER and the NE (arrow). Scale bars: 15 µm.
78 Figure 19: In vitro transport assay to the ER and NE using VAPB and PTP1B expressed in a complex with TRC40. Transport reactions of a putative TA-protein together with TRC40 to the ER membranes or NE, were performed directly on the coverslips. One day prior to the transport assay, HeLa P4 cells were seeded on coverslips. After digitonin-permeabilization of the cells, the transport reaction was set up. The amount of TRC40/TA-protein-complexes used per import reaction was for VAPB 2.7 µg (0.6 µM) and for PTP1B 1 µg (1 µM). The reaction was completed by addition of cytosol and ATP-regenerating system to the protein and the mixture was added to the cells. Transport was performed at 37 °C for one hour and an additional control reaction at 4 °C was prepared. Cy3-BSA-NLS served as a general control for the assay and should localize to the nucleoplasm as a soluble cargo protein. The fluorescent signal in the green channel (α-opsin) indicates integration of the protein into the ER and perhaps the NE. Scale bars: 10 µm.