Newly synthesized integral INM proteins are integrated into the ER membrane and afterwards traverse through the ONM to the INM via the NPC. To pass the central channel of the NPC, the nucleoplasmic domains of INM proteins would have to extend to the central channel from the membrane surface (Antonin et al., 2011). A size dependency of ~60 kDa for the nucleoplasmic domain for the passage through the central channel has been demonstrated. (Ohba et al., 2004; Soullam and Worman, 1995). Peripheral channels adjacent to the pore complex might also allow passage of membrane proteins (Maimon et al., 2012).
Four models of INM targeting have been proposed: transport-factor mediated targeting, localization based on diffusion and retention, targeting via an INM signal sequence (sorting-motif) and transport through the NPC with the help of FG-motifs (Katta et al., 2014; Ungricht et al., 2015). In the transport-factor mediated model (Figure 7), INM proteins containing a NLS interact with a nuclear transport factor in the cytoplasm. The
LBR MAN1emerin Lap2ß
nuclear lamina cytoplasm
nucleoplasm ER
ONM
INM NPC
cargo-transport factor complex then passes through the NPC either through the central channel or via the peripheral channel. After reaching the nucleus, the transport factor is released from the cargo by Ran-GTP (Katta et al., 2014; Laba et al., 2014).
Figure 7. Major models of membrane protein trafficking to the INM.
The transport factor-mediated model uses similar principles of transport as those established for soluble proteins. Cargo proteins containing an NLS bind to transport factors and are transported through interaction with nucleoporins of the central channel. After reaching the nucleus, dissociation of the cargo-transport factor is mediated by RanGTP. The diffusion and retention model suggests that INM proteins laterally diffuse through the peripheral channel of the NPC, from the ER via the ONM to the INM. The proteins are retained at the INM by interacting with nuclear lamins or chromatin.
It was previously reported that the yeast LEM-domain containing proteins Heh1 and Heh2 require active transport using karyopherin-a (Kap60) and karyopherin-b (Kap95), and also the RanGTPase cycle (King et al., 2006). An NLS was identified in Heh2 that binds to karyopherins, which was important for its INM localization (Liu et al., 2010; Meinema et al., 2011). Many of the INM proteins may contain a putative NLS in their extraluminal domains, suggesting that this could be a widely used mechanism for INM targeting (Lusk et al., 2007).
A possibility to consider with this targeting mechanism is whether INM proteins extend their NLSs through sideward openings of peripheral channel to provide a handle for transport-factor mediated translocation through the central NPC channel (Turgay et al., 2010).
Therefore, the functional and mechanistic contributions of these NLSs to the INM protein targeting process needs to be further investigated. A variant of transport factor mediated model was also described suggesting that INM proteins bind to soluble NLS-containing cargoes and ‘piggyback’ on their transport factor-mediated transport to reach the INM (Gardner et al., 2011).
In the diffusion and retention model (Figure 7), INM proteins diffuse from the ER to the ONM, and from the ONM to the INM through the peripheral channels of the NPC. After reaching the INM, the proteins are retained by tethering to nuclear components like lamins
NLS transport factor
cargo
Ran GTP
nucleoplasm ER
cytoplasm
Diffusion and retention Transport factor mediated
cargo
cytoplasm
nucleoplasm lamins
ER lamin or chromatin binding domain
1995). This peripheral channel imposes a size limit of less than 40 kDa for the extraluminal domain of the INM proteins (Soullam and Worman, 1995). In line with this model, photobleaching studies performed on several INM proteins showed rapid diffusion from the ER to the INM (Ellenberg et al., 1997; Ostlund et al., 1999; Shimi et al., 2004; Ungricht et al., 2015; Wu et al., 2002; Zuleger et al., 2011). The mobility of the tested proteins was reduced at the INM compared to the ER, suggesting that they are associated with relatively immobile lamins or chromatin (Ungricht et al., 2015; Zuleger et al., 2011).
The signal sequence or sorting motif model relies on small, charged motifs on the INM proteins that are recognized by membrane-associated, short isoforms of karyopherins (for example, Importin a-16). The transport occurs through the peripheral channel of the NPC and after reaching the nucleus, the protein release is stimulated by Nup50/Nup2, as reported for the yeast protein Heh2 (Braunagel et al., 2004; Braunagel et al., 2007; Saksena et al., 2004; Saksena et al., 2006). The fourth model is based on a systematic study performed on 15 different INM proteins suggesting that many INM proteins are enriched in FG-repeats that possibly allow for direct translocation of these proteins through the NPC and could use multiple overlapping pathways to reach INM (Katta et al., 2014; Zuleger et al., 2011). The transport models as well as the membrane-insertion pathways established for some well-characterized INM proteins are listed in Table 2.
Table 2. Models of targeting of well-characterized INM proteins.
Protein ER membrane
LBR co-translational diffusion and retention, mobility dependent on
Lap2b post-translational diffusion and retention 1 (Furukawa et al., 1995;
Furukawa et al., 1998;
Ohba et al., 2004;
Zuleger et al., 2011) emerin post-translational diffusion and retention,
mobility requires ATP
1 (Berk et al., 2013a;
Zuleger et al., 2011) Man1 co-translational diffusion and retention 2 (Wu et al., 2002) Heh1
Protein ER membrane insertion
Nuclear import machinery
Number of TMDs
References
Heh2 (yeast)
co-translational transport factor mediated
2 (King et al., 2006; Liu et al., 2010; Meinema et al., 2011)