Characterization of Wiskott^aldrich Syndrome(was)mutants Using Saccharomyces Cerevisiae

Abstract

Wiskott-Aldrich syndrome (WAS) is caused by alterations in the WAS protein

(WASP), and 80% of the missense mutations are located in the WH1 domain, the

region essential for interaction with the WASP-interacting protein (WIP). It has

been suggested that loss of WASP-WIP interaction is causal to the disease. Las17p

(yeast WASP) is essential for growth at 37 1C. The growth defect of the las17D

strain can be suppressed by the expression of human WASP together with WIP.

Using the las17D strain, we have analyzed 52 missense mutations in the gene

encoding WASP and found that 13 of these mutant proteins were unable to

suppress the growth defect of the las17D strain. The majority of these 13 mutations

cause the classic WAS in humans and are located within the WH1 domain, while

none of the 12 mutations outside the WH1 domain abolished the activity ofWASP

in Saccharomyces cerevisiae cells. This suggests that some of the mutations (13 out

of 40) in the WH1 domain cause the syndrome in humans by perturbing the

WASP-WIP complex formation, while the rest of the mutations cause the

syndrome without affecting the WASP-WIP complex formation, but may affect

the activity of the complex.

Introduction

Wiskott-Aldrich syndrome (WAS) is an X-linked genetic

disease caused by recessive mutations in the gene encoding

the WAS protein (WASP) (Derry et al., 1994). WASP is a

proline-rich protein and has a modular structure organized

into several domains: a WASP homology (WH1) domain, a

basic region, a GTPase-binding domain, a proline-rich

sequence and the Verprolin homology, Cofilin homology

and the Acidic region (VCA) domain (Takenawa & Suetsugu,

2007). The VCA domain of WASP activates the Arp2/3

complex to regulate actin polymerization (Rohatgi et al.,

1999). WASP is expressed predominantly in hematopoietic

cells while its homologue N-WASP (neural), with an additional

V domain, is expressed ubiquitously (Miki et al.,

1996). Both N-WASP and WASP have been shown to

interact with Cdc42 (Symons et al., 1996) and many

cytoskeletal proteins including WASP-interacting protein

(WIP) (Ramesh et al., 1997). WASP and N-WASP adopt an

inactive conformation (Kim et al., 2000) that can be

activated by Cdc42 (Higgs & Pollard, 2000; Rohatgi et al.,

2000). Recently, it has been proposed that activation of NWASP

by Cdc42 is mediated by the transducer of Cdc42

activation (Toca-1) (Ho et al., 2004), and it was found that

Toca-1 and not Cdc42 relieves the autoinhibition of WASP

(Lim et al., 2007). Las17p is the yeast homologue of

mammalian WASP (Li, 1997; Naqvi et al., 1998), and

deletion of the gene encoding Las17p results in a Saccharomyces

cerevisiae strain with cytoskeletal and growth defects;

the las17D strains are unable to grow at elevated temperatures

and have defects in fluid-phase endocytosis and actin

patch polarization at both permissive and restrictive temperatures

(Li, 1997; Naqvi et al., 1998).

In mammalian cells, the majority of WASP molecules are

present in a complex with WIP (Sasahara et al., 2002; de la

Fuente et al., 2007).Most of the missense mutations (80%