Lamina-associated Polypeptide 2-α Forms Homo-trimers via Its C Terminus, and Oligomerization Is Unaffected by a Disease-causing Mutation

Luc Snyers,Sylvia Vlcek,Thomas Dechat,Darko Skegro,Barbara Korbei,Andreas Gajewski,Olga Mayans,Christian Schöfer,Roland Foisner

doi:10.1074/jbc.m605782200

Abstract

The nucleoplasmic protein, Lamina-associated polypeptide (LAP) 2alpha, is one of six alternatively spliced products of the LAP2gene, which share a common N-terminal region. In contrast to the other isoforms, which also share most of their C termini, LAP2alpha has a large unique C-terminal region that contains binding sites for chromatin, A-type lamins, and retinoblastoma protein. By immunoprecipitation analyses of LAP2alpha complexes from cells expressing differently tagged LAP2alpha proteins and fragments, we demonstrate that LAP2alpha forms higher order structures containing multiple LAP2alpha molecules in vivo and that complex formation is mediated by the C terminus. Solid phase binding assays using recombinant and in vitro translated LAP2alpha fragments showed direct interactions of LAP2alpha C termini. Cross-linking of LAP2alpha complexes and multiangle light scattering of purified LAP2alpha revealed the existence of stable homo-trimers in vivo and in vitro. Finally, we show that, in contrast to the LAP2alpha-lamin A interaction, its self-association is not affected by a disease-linked single point mutation in the LAP2alpha C terminus.

Highlights

In addition to the lamins, the nuclear lamina contains a number of integral membrane proteins of the inner nuclear membrane, the best characterized of which are the Lamin B receptor, Lamina-associated polypeptide (LAP),4 and the three LEM domain-containing proteins LAP2␤, emerin and MAN1 [5, 6]
The C Terminus of LAP2␣ Is Involved in the Formation of Oligomeric Complexes in Vivo—Our previous studies revealed the formation of stable, chromatin-associated LAP2␣ structures during early nuclear assembly stages [14, 17], indicating oligomerization of the protein
Both constructs reacted with polyclonal antibodies against LAP2␣ C terminus) and antibodies against red fluorescent protein, whereas a monoclonal antibody directed against the N-terminal region of LAP2␣ that is common to all LAP2 isoforms recognized only monomeric red fluorescent protein 1 (mRFP1)-LAP2␣ plus the endogenous protein

Summary

Introduction

In addition to the lamins, the nuclear lamina contains a number of integral membrane proteins of the inner nuclear membrane, the best characterized of which are the Lamin B receptor, Lamina-associated polypeptide (LAP),4 and the three LEM domain-containing proteins LAP2␤, emerin and MAN1 [5, 6]. By immunoprecipitation analyses of LAP2␣ complexes from cells expressing differently tagged LAP2␣ proteins and fragments, we demonstrate that LAP2␣ forms higher order structures containing multiple LAP2␣ molecules in vivo and that complex formation is mediated by the C terminus.

Results

Conclusion