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

Abstract

Protein targeting to the inner nuclear membrane (INM) is one of the least understood protein targeting pathways. INM proteins are important for chromatin organization, nuclear morphology and movement, and meiosis, and have been implicated in human diseases. In opisthokonts, one mechanism for INM targeting is transport factor-mediated trafficking, in which nuclear localization signals (NLSs) function in nuclear import of transmembrane proteins. To explore whether this pathway exists in plants, we fused the SV40 NLS to a plant ER tail-anchored protein and showed that the GFP-tagged fusion protein was significantly enriched at the nuclear envelope (NE) of leaf epidermal cells. Airyscan subdiffraction limited confocal microscopy showed that this protein displays a localization consistent with an INM protein. Nine different monopartite and bipartite NLSs from plants and opisthokonts, fused to a chimeric tailanchored membrane protein, were all sufficient for NE enrichment, and both monopartite and bipartite NLSs were sufficient for trafficking to the INM. Tolerance for different linker lengths and protein conformations suggests that INM trafficking rules might differ from those in opisthokonts. The INM proteins developed here can be used to target new functionalities to the plant nuclear periphery. This article has an associated First Person interview with the first author of the paper.

Casting a Wider Net: Differentiating between Inner Nuclear Envelope and Outer Nuclear Envelope Transmembrane Proteins

“…Since the Airyscan microscopy method does not have enough resolution to visually distinguish between the ONM and INM, line scans of the NE were used to determine co-localization of the two proteins (Figure 3a,b). However, even line scans are limited by the overall resolution of the system, therefore several statistical analyses had to performed on the line scan results before providing satisfying conclusions [63].…”

Regulation and Physiological Significance of the Nuclear Shape in Plants

“…KAKU4-GFP overexpression causes nuclear membrane growth and NE deformation in whole seedlings of Arabidopsis and leaves of N. tabacum and N. benthamiana ( Goto et al, 2014 ). Indeed, care is required to interpret the phenotypes caused by the overexpression of GFP-fused proteins, as they can impose artificial effects ( Groves et al, 2019 ). However, KAKU4 fused to another tag, FLAG , also causes NE deformation ( Goto et al, 2014 ).…”