New N-RAP-binding partners alpha-actinin, filamin and Krp1 detected by yeast two-hybrid screening: implications for myofibril assembly.

Shajia Lu,Robert Horowits,Amy H Herrera,Stefanie L Carroll,Bradford Ozanne

doi:10.1242/jcs.00425

Abstract

N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly. To discover more about the role of N-RAP in myofibril assembly, we used the yeast two-hybrid system to screen a mouse skeletal muscle cDNA library for proteins capable of binding N-RAP in a eukaryotic cell. From yeast two-hybrid experiments we were able to identify three new N-RAP binding partners: alpha-actinin, filamin-2, and Krp1 (also called sarcosin). In vitro binding assays were used to verify these interactions and to identify the N-RAP domains involved. Three regions of N-RAP were expressed as His-tagged recombinant proteins, including the nebulin-like super repeat region (N-RAP-SR), the N-terminal LIM domain (N-RAP-LIM), and the region of N-RAP in between the super repeat region and the LIM domain (N-RAP-IB). We detected significant alpha-actinin binding to N-RAP-IB and N-RAP-LIM, filamin binding to N-RAP-SR, and Krp1 binding to N-RAP-SR and N-RAP-IB. During myofibril assembly in cultured chick cardiomyocytes, N-RAP and filamin appear to co-localize with alpha-actinin in the earliest myofibril precursors found near the cell periphery, as well as in the nascent myofibrils that form as these structures fuse laterally. In contrast, Krp1 is not localized until late in the assembly process, when it appears at the periphery of myofibrils that appear to be fusing laterally. The results suggest that sequential recruitment of N-RAP binding partners may serve an important role during myofibril assembly.

Highlights

N-RAP is an actin binding LIM protein expressed in skeletal and cardiac muscle tissues (Luo et al, 1997)
These regions are the myotendinous junctions in skeletal muscle and the intercalated disks in heart muscle, and ultrastructural data show that N-RAP co-localizes with the terminal actin bundles that link the myofibrils to the membrane in these regions (Herrera et al, 2000; Zhang et al, 2001)
On the basis of the localization and binding affinities of N-RAP, we proposed that N-RAP serves as a link between the terminal actin of the myofibril and the protein complexes at the cell membrane (Luo et al, 1999; Luo et al, 1997)

Summary

Introduction

N-RAP is an actin binding LIM protein expressed in skeletal and cardiac muscle tissues (Luo et al, 1997). Additional evidence suggests that muscle myosin may separately assemble into bipolar thick filaments that are oriented and incorporated into the nascent myofibrils by their interactions with titin filaments (Holtzer et al, 1997; Rudy et al, 2001; Schultheiss et al, 1990). Nebulette, another protein with nebulin-related repeats, appears to associate with myofibril precursors early during assembly; nebulette is expressed only in cardiac muscle and remains associated with mature Z-lines (Moncman and Wang, 1995; Moncman and Wang, 1999). N-RAP is found in all the myofibril precursors in cultured cardiomyocytes, it is not found in mature sarcomeres (Carroll and Horowits, 2000; Luo et al, 1997) and is expressed in both skeletal and cardiac muscle (Luo et al, 1997; Zhang et al, 2001)

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