Identification of the evolutionarily conserved nuclear envelope proteins Lem2 and MicLem2 in Tetrahymena thermophila.

Masaaki Iwamoto,Yasushi Hiraoka,Hiroko Osakada,Tokuko Haraguchi,Chie Mori,Yasuhiro Fukuda

doi:10.1016/j.gene.2019.100006

Abstract

Lem2 family proteins, i.e. the LAP2-Emerin-MAN1 (LEM) domain-containing nuclear envelope proteins, are well-conserved from yeasts to humans, both of which belong to the Opisthokonta supergroup. However, whether their homologs are present in other eukaryotic phylogenies remains unclear. In this study, we identified two Lem2 homolog proteins, which we named as Lem2 and MicLem2, in a ciliate Tetrahymena thermophila belonging to the SAR supergroup. Lem2 was localized to the nuclear envelope of the macronucleus (MAC) and micronucleus (MIC), while MicLem2 was exclusively localized to the nuclear envelope of the MIC. Immunoelectron microscopy revealed that Lem2 in T. thermophila was localized to both the inner and outer nuclear envelopes of the MAC and MIC, while MicLem2 was mostly localized to the nuclear pores of the MIC. Molecular domain analysis using GFP-fused protein showed that the N-terminal and luminal domains, including the transmembrane segments, are responsible for nuclear envelope localization. During sexual reproduction, enrichment of Lem2 occurred in the nuclear envelopes of the MAC and MIC to be degraded, while MicLem2 was enriched in the nuclear envelope of the MIC that escaped degradation. These findings suggest the unique characteristics of Tetrahymena Lem2 proteins. Our findings provide insight into the evolutionary divergence of nuclear envelope proteins.

Highlights

The nuclear envelope (NE) is a cell structure that physically and functionally separates genomic DNA from the cytoplasm
To identify its homologous proteins in the ciliate T. thermophila, which belongs to the Alveolata group (Fig. 1B), we searched for proteins possessing the MSC domain in the NCBI protein database; the MSC domain sequences of human Lem2, human Man1, C. elegans Lem2, S. pombe Lem2, S. pombe Man1, S. cerevisiae Heh2p, and S. cerevisiae Src1p were used as a query sequence in BLAST
One of the characteristic features of LEM domain proteins is the presence of an LEM domain, which is a bi-helical motif, in the N-terminus (Laguri et al, 2001; Cai et al, 2001)

Summary

Introduction

The nuclear envelope (NE) is a cell structure that physically and functionally separates genomic DNA from the cytoplasm. The NE is composed of the outer and inner nuclear membranes (ONM and INM, respectively), which are connected to the pore membrane (reviewed in Goldberg and Allen, 1995; De Magistris and Antonin, 2018). In addition to this conserved structure, the nuclear lamina, a protein meshwork composed of lamins (type V intermediate filament proteins), is underneath the INM only in metazoan (reviewed in de Leeuw et al, 2018). The INM contains INM-specific integral membrane proteins; the ONM continues into the endoplasmic reticulum (ER). Several hundred putative transmembrane proteins have been identified in mammalian cells through proteomics analysis (Schirmer et al, 2003; Korfali et al, 2012; de Las Heras et al, 2013)

Methods

Results

Conclusion