The Chediak-Higashi Protein Interacts with SNARE Complex and Signal Transduction Proteins

Velizar T Tchernev,A Madan Kumar,Jonathan M Rothberg,Ying Li,Loic Giot,Vishnu S Mishra,Krishnan Nandabalan,Frederick S Southwick,John C Detter,Margaret R Wallace,Stephen F Kingsmore,Traci A Mansfield

doi:10.1007/bf03402003

Velizar T Tchernev, A Madan Kumar + Show 10 more

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https://doi.org/10.1007/bf03402003

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Abstract

Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay. Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II). On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition.

Highlights

Lysosomes have unique and fascinating abnormalities in Chediak–Higashi syndrome (CHS), a primary immunodeficiency disease
Fourteen lysosomal trafficking regulator (LYST) cDNA fragments covering the coding domain were cloned into activationand DNA binding-domain vectors
Keratinocyte, fetal brain, fetal liver, and fetal kidney cDNA libraries were screened for LYST-interacting proteins (LIPs)

Summary

Introduction

Lysosomes have unique and fascinating abnormalities in Chediak–Higashi syndrome (CHS), a primary immunodeficiency disease. They are greatly enlarged, limited to the perinuclear region, lack critical cargo proteins, and are dilatory in heterotypic membrane fusion [1,2,3]. The clinical features of CHS of recurrent infections, albinism, and bleeding diathesis reflect these defects. For example, result from secretory granule protein missorting and impaired leukocyte exocytosis [4,5,6]. Chediak–Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. Five of the interactions were confirmed by an in vitro binding assay

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