Distinctive Properties of the Nuclear Localization Signals of Inner Nuclear Membrane Proteins Heh1 and Heh2

Abstract

SUMMARY Targeting of Endoplasmic Reticulum (ER)-synthesized membrane proteins to the Inner Nuclear Membrane (INM) has long been explained by the ‘diffusion-retention model’. However, several INM proteins contain non-classical Nuclear Localization Signal (NLS) sequences that, in a few instances, have been shown to promote importin α/β– and Ran–dependent translocation to the INM. Here, using structural and biochemical methods, we show that yeast INM proteins Heh2 and Src1/Heh1 contain bipartite import sequences that associate intimately with the minor NLS-binding pocket of yeast importin α and unlike classical NLSs efficiently displace the IBB-domain in the absence of importin β. In vivo, the intimate interactions at the minor NLS-binding pocket make the h2NLS highly efficient at recruiting importin α at the ER and drive INM localization of endogenous Heh2. Thus, h1/h2NLSs delineate a novel class of super-potent, IBB-like membrane protein NLSs, distinct from classical NLSs found in soluble cargos, and of general interest in biology.

Divergent Evolution of Nuclear Localization Signal Sequences in Herpesvirus Terminase Subunits

“…A similar enthalpy release was measured in a control experiment where the classical SV40-NLS was injected against ⌬IBB-importin ␣ under identical experimental conditions (Fig. 4C), which gave equilibrium dissociation constants K d1 ϭ 1.5 Ϯ 0.3 M and K d2 ϭ 9.7 Ϯ 1.2 M. It should be pointed out that the equilibrium binding constants reported here are significantly lower than those measured using fluorescence depolarization (79) or surface-immobilized NLSs (80) but nonetheless consistent with published ITC studies that employed short NLS peptides (47,75,76,81). This is explained by the high solubility and charge of NLS peptides and their tendency to remain in solution as solvated ions.…”

A nuclear localization signal targets tail-anchored membrane proteins to the inner nuclear envelope in plants

“…The yeast INM protein Heh2 remains the most well studied INM protein from the perspective of trafficking. The Heh2 NLS-importin interaction has been mapped, and the Heh2 bipartite NLS has been determined to be atypical in that it strongly competes off the autoinhibitory IBB domain of importin α (Lokareddy et al, 2015). The Heh2 NLS also works in nuclear import of membrane proteins in mammalian cells, and conversely, the closely related NLS of Fig.…”

“…The bipartite NLS of yeast Heh2 (and Heh1) strongly associates with the minor NLS-binding pocket of importin α in a different manner from classical NLS binding, and this difference likely plays a role in making the Heh2 NLS more effective for membrane protein nuclear import in yeast (Kralt et al, 2015;Lokareddy et al, 2015). The above data suggest that re-targeting from the ER to the NE in N. benthamiana functions similarly when driven by the bipartite Heh2 NLS and by the classical SV40 NLS (compare Fig.…”

An ESCRT-LEM domain inner nuclear membrane protein surveillance system is poised to directly monitor the integrity of the nuclear envelope barrier and nuclear transport system

“…Heh1 and Heh2 contain an N-terminal helix-extension-helix (heh) motif (the LEM domain), followed by an INM targeting sequence that (at least in the case of Heh2 but likely also Heh1 (King et al, 2006;Lokareddy et al, 2015)) includes an NLS and a ~200 amino acid unstructured region that are both required for INM targeting (Meinema et al, 2011). They both also contain a second nuclear-oriented domain, which likely folds into a winged helix (WH)(also called MAN1/Src1-C-terminal homology domain or MSC; (Caputo et al, 2006)); this domain is also well conserved through evolution (Mans et al, 2004;Mekhail et al, 2008).…”