Lst4, the yeast Fnip1/2 orthologue, is a DENN-family protein.

Angela Pacitto,Beata K Blaszczyk,Svetlana Dokudovskaya,Nianshu Zhang,Tom L Blundell,Ravi K Nookala,Louise H Wong,David B Ascher,Tim P Levine

doi:10.1098/rsob.150174

Abstract

The folliculin/Fnip complex has been demonstrated to play a crucial role in the mechanisms underlying Birt–Hogg–Dubé (BHD) syndrome, a rare inherited cancer syndrome. Lst4 has been previously proposed to be the Fnip1/2 orthologue in yeast and therefore a member of the DENN family. In order to confirm this, we solved the crystal structure of the N-terminal region of Lst4 from Kluyveromyces lactis and show it contains a longin domain, the first domain of the full DENN module. Furthermore, we demonstrate that Lst4 through its DENN domain interacts with Lst7, the yeast folliculin orthologue. Like its human counterpart, the Lst7/Lst4 complex relocates to the vacuolar membrane in response to nutrient starvation, most notably in carbon starvation. Finally, we express and purify the recombinant Lst7/Lst4 complex and show that it exists as a 1 : 1 heterodimer in solution. This work confirms the membership of Lst4 and the Fnip proteins in the DENN family, and provides a basis for using the Lst7/Lst4 complex to understand the molecular function of folliculin and its role in the pathogenesis of BHD syndrome.

Highlights

Birt–Hogg–Dube (BHD) syndrome is a rare autosomal-dominantly inherited disorder which predisposes patients to benign tumours of the hair follicle, lung cysts that give rise to pneumothoraces and kidney tumours [1]
Flcn has been shown to be involved in numerous signalling pathways, including the mechanistic target of rapamycin complex 1 pathway [3,4,5], energy sensing through AMP-activated protein kinase (AMPK) [3,6], the transforming growth factor b pathway [7], autophagy regulation [8,9] and Wnt signalling [10] among others, though a precise understanding of its role at the molecular level remains to be achieved
In order to confirm that Lst4 and Fnip1/2 belong to the differentially expressed in normal and neoplastic (DENN) family, we decided to pursue structure determination of this protein

Summary

Introduction

Birt–Hogg–Dube (BHD) syndrome is a rare autosomal-dominantly inherited disorder which predisposes patients to benign tumours of the hair follicle (fibrofolliculomas), lung cysts that give rise to pneumothoraces and kidney tumours [1] This disorder arises from germline mutations in the FLCN gene [2], and much effort over the past decade has gone into unravelling the molecular function of its protein product, folliculin (Flcn). Flcn is known to have two paralogous binding partners, the Flcn interacting partners Fnip and Fnip, which interact independently with Flcn [3,11,12] This Flcn/Fnip (either Flcn/ Fnip or Flcn/Fnip2) complex has been recently reported to be involved in amino acid sensing through regulation of the Rag GTPases at the lysosomal membrane and controlling signalling through mTORC1 [13,14].

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Results

Conclusion