3.  Do the identified proteins colocalize with Mlp1p?

Colocalization experiments:

A.  Do Mlp1p and the protein colocalize when visualized by immunoelectron microscopy?

Wildtype cells would be fixed and sectioned, then exposed to antibody against Mlp1p and a second antibody against the interacting protein.  Secondary antibodies with varying sizes of gold conjugated to them would be allowed to react with the primary antibody.  The cells and the location of the different gold particles would be visualized using transmission electron microscopy.  If the two proteins were interacting with each other, the two sizes of gold particles would be located near each other.  A negative control would be to treat the cells with irrelevant antibody before adding secondary antibody to determine the extent of any nonspecific interactions that occur. 

B.  Do Mlp1p and the protein cofractionate?

Yeast cells would be fractionated to give nuclei, nuclear envelope, and NPCs.  These fractions would be run on SDS-PAGE gels.  Western blots would be done to detect both Mlp1p protein and the interacting protein and the presence of each in the various fractions would be compared.  If the proteins cofractionate, this data would support the mass spec data. 

Controls:  blot with antibodies against proteins known to be found only in each of the compartments to monitor that each fraction is truly what it is intended to be. 

Determining whether the proteins localize to each other in the cell provides confirmation of the mass spectrometry data. 

These experiments will help to define interactions between Mlp1p and other proteins that are involved in nuclear transport.  Once these proteins are identified, further analysis can be done to determine how Mlp1p is specifically involved in nuclear transport.  By determining the function of proteins that interact with Mlp1p, the existence of similar interactions between Tpr and homologous interacting proteins could be investigated in the mammalian system.

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