MISTIC-fusion proteins as antigens for high quality membrane protein antibodies.

Lack of high-quality antibodies against transmembrane proteins is a widely recognized hindrance in biomedical and cell biological research. Here we present a robust pipeline for the generation of polyclonal antibodies employing full-length membrane proteins as immunogens to overcome this "antibody bottleneck". We express transmembrane proteins fused to a MISTIC fragment that enhances expression of eukaryotic membrane proteins in E. coli. Purified membrane proteins are used as immunogen for rabbit injection employing standard immunizing protocols. The raised antibodies against membrane proteins of the endoplasmic reticulum and the nuclear envelope, which we use as test cases, function in a wide range of applications and are superior to ones produced against soluble domains as immunogens.

Nup153 Recruits the Nup107-160 Complex to the Inner Nuclear Membrane for Interphasic Nuclear Pore Complex Assembly.

In metazoa, nuclear pore complexes (NPCs) are assembled from constituent nucleoporins by two distinct mechanisms: in the re-forming nuclear envelope at the end of mitosis and into the intact nuclear envelope during interphase. Here, we show that the nucleoporin Nup153 is required for NPC assembly during interphase but not during mitotic exit. It functions in interphasic NPC formation by binding directly to the inner nuclear membrane via an N-terminal amphipathic helix. This binding facilitates the recruitment of the Nup107-160 complex, a crucial structural component of the NPC, to assembly sites. Our work further suggests that the nuclear transport receptor transportin and the small GTPase Ran regulate the interaction of Nup153 with the membrane and, in this way, direct pore complex assembly to the nuclear envelope during interphase.