![]() ![]() A variant of this model, based on the yeast protein Mps3, proposed that INM proteins bind to soluble import substrates and “piggyback” on their transport receptor–mediated nuclear import ( Gardner et al., 2011). Here, translocation is believed to involve the cytoplasmic domain of INM proteins to reach into the central channel of the NPC, implying a continuous open path for membrane proteins through the walls of the NPC channel ( Meinema et al., 2011). This model is mostly based on studies on the yeast INM proteins Heh1p and Heh2p, which require a functional RanGTPase system as well as importin α and β1 for the INM protein targeting process ( King et al., 2006 Meinema et al., 2011). More recent studies have suggested a different model, termed receptor-mediated translocation in analogy to the well characterized transport mechanism of soluble nuclear proteins ( King et al., 2006 Meinema et al., 2011). This model has been supported by early studies on different INM proteins ( Powell and Burke, 1990 Smith and Blobel, 1993 Soullam and Worman, 1995 Ellenberg et al., 1997 Yang et al., 1997) and does not require active or receptor-mediated translocation of INM proteins across the NPC. Enrichment in the INM over the ER/ONM then occurs by interaction with nuclear binding partners such as lamins or chromatin, which would be required to retain INM proteins in the nucleus. This peripheral channel would impose a size constraint on the cytoplasmic domain of INM proteins ( Soullam and Worman, 1995 Theerthagiri et al., 2010 Antonin et al., 2011). In the diffusion retention model, the translocation between the ONM and INM is believed to occur by undirected passive diffusion through a narrow ∼10-nm-diameter peripheral channel between the NPC and nuclear membrane ( Reichelt et al., 1990 Beck et al., 2004). A key mechanistically controversial step is INM protein translocation from the ONM to the INM at the NPC, for which different models have been proposed depending on the type of INM protein and organism studied. Newly synthesized INM proteins are inserted into the ER membrane from where they move laterally by diffusion through ER cisternae and tubules to the ONM. Collectively, our data support a diffusion retention model of INM protein transport in mammalian cells. These predictions were confirmed with targeted validation experiments on the functional requirements of nucleoporins and nuclear lamins. Modeling of the phenotypes of genes involved in transport of these INM proteins predicted that it critically depended on the number and permeability of nuclear pores and the availability of nuclear binding sites, but was unaffected by depletion of most transport receptors. These reporters allowed us to characterize the kinetics of INM targeting and establish a mathematical model of this process and enabled us to probe its molecular requirements in an RNA interference screen of 96 candidate genes. ![]() Here, we established a new reporter that allows real-time imaging of membrane protein transport from the ER to the INM using Lamin B receptor and Lap2β as model INM proteins. Targeting of inner nuclear membrane (INM) proteins is essential for nuclear architecture and function, yet its mechanism remains poorly understood. ![]()
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