2. What proteins interact with Mlp1p?
If domains of Mlp1p that are important for nuclear import but not for association with the nuclear pore complex or cellular localization are identified, it is likely that these domains could interact with proteins involved in the transport process.
It is known that the C-terminal region of Mlp1p, like that of Tpr, is free and may be able to interact with various proteins.
It has been shown that in Xenopus, Tpr binds importin ß.
Previous data suggests that Mlp1p is involved in protein import, which involves a cargo protein translocated through the NPC in association with importin α and importin ß.
Therefore, it could be tested whether Mlp1p binds the yeast karyopherin ß1 by coimmunoprecipitation. Epitope tagged MLP1 constructs could be made by adding, for example, a Myc-tag to one end of the protein by a PCR method. These constructs would be ligated into a vector and transformed into a mlp1Δ strain. Proper localization of the fusion protein would have to be monitored. For the coimmunoprecipitation, extracts from the following strains of yeast would be used:
mlp1Δ + empty vector
mlp1Δ + epitope tagged MLP1 construct
Anti-myc antibody would be added to these extracts, then protein A sepharose beads would be added. The protein pulled down would be run on an SDS-PAGE gel, and a Western blot would be done to probe for karyopherin ß1. The presence of a band at the appropriate molecular weight for karyopherin ß1 in in the strain with the tagged construct but not in the empty vector would indicate that the two proteins interacted. However, this does not show that the interaction is necessarily direct.
In order to obtain support that the interaction (if present) is direct, purified proteins could be obtained and used in pulldown experiments.
3. What proteins are present in complexes associated with Mlp1p and Tpr?
To identify further proteins that interact with Mlp1p and may have a role in the nuclear transport of proteins, an pulldown experiment using mass spectrometry will be done. This type of experiment has been done by Allen et al. (2001) in order to identify nucleoporin interacting proteins.
The same approach could be used by making a GST construct of Mlp1p. A GST construct of Mlp1p would be made by ligating genomic DNA into the vector pGEX-2TK. The protein would then be expressed in E. coli and isolated using glutathione-Sepharose beads. These beads with the Mlp1p construct attached are incubated in yeast extract containing approximately 10 mg of protein. The beads and attached complexes are then washed, and the protein complexes associated with the beads are extracted by the addition of salt or SDS. The proteins are then run on SDS-PAGE gels and visualized with Coomassie Blue.
As a negative control, glutathione-Sepharose beads with only glutathione-S-transferase should be used to compare what proteins are being pulled down by the GST portion of the fusion protein.
Once the proteins are visualized on the gel, bands that are present in the Mlp1-GST lane but not in the GST-only lane will be excised, destained, and digested with trypsin. The peptides will be extracted from the gel and analyzed by MALDI-TOF mass spectrometry. If two proteins occupy the same band on the SDS-PAGE gel, the proteins could be further separated and analyzed by liquid chromatography mass spectroscopy. The masses obtained will be compared against a database of theoretical tryptic digests of all yeast proteins such as MS-Fit to determine the protein identity.
A similar approach could be used to determine proteins that interact with mammalian Tpr.
The validity of the data obtained from the mass spectrometry experiment could be verified by doing coimmunoprecipitations using tagged Mlp1 and cell extracts.
It would be expected that nucleoporins would interact with both Mlp1 and Tpr. Other proteins involved in nuclear transport might also interact. Again, this experiment does not imply a direct interaction between Mlp1 or Tpr and the identified proteins. However, the interactions could be further explored using purified proteins to determine if the interactions were direct.
The domains of Mlp1p/Tpr important for such identified interactions could be assayed by using constructs with various portions of the proteins deleted and doing pulldown assays. GST constructs of the Mlp1p and Tpr proteins could be made and expressed in E. coli, then bound to glutathione-Sepharose beads. These beads could be incubated with the appropriate expressed proteins (those identified by mass spec), and the resulting complexes analyzed using SDS-PAGE and Western blotting. The control for these experiments would be glutathione-S-transferase beads incubated with the appropriate cell extract.