Requirement of the spindle pole body for targeting and/or tethering proteins to the inner nuclear membrane

Abstract

Appropriate targeting of inner nuclear membrane (INM) proteins is important for nuclear function and architecture. To gain new insights into the mechanism(s) for targeting and/or tethering peripherally associated proteins to the INM, we screened a collection of temperature sensitive S. cerevisiae yeast mutants for defects in INM location of the peripheral protein, Trm1-II-GFP. We uncovered numerous genes encoding components of the Spindle Pole Body (SPB), the yeast centrosome. SPB alterations affect the localization of both an integral (Heh2) and a peripheral INM protein (Trm1-II-GFP), but not a nucleoplasmic protein (Pus1). In wild-type cells Trm1-II-GFP is evenly distributed around the INM, but in SPB mutants, Trm1-II-GFP mislocalizes as a spot(s) near ER-nucleus junctions, perhaps its initial contact site with the nuclear envelope. Employing live cell imaging over time in a microfluidic perfusion system to study protein dynamics, we show that both Trm1-II-GFP INM targeting and maintenance depend upon the SPB. We propose a novel targeting and/or tethering model for a peripherally associated INM protein that combines mechanisms of both integral and soluble nuclear proteins, and describe a role of the SPB in nuclear envelope dynamics that affects this process.