SOLUSYKLI: Lähdetietoa ESCRT-0 ja ESCRT-1 väliltä (artikkelit vuosilta 2006 ja 2017)

torsdag 17 maj 2018

Lähdetietoa ESCRT-0 ja ESCRT-1 väliltä (artikkelit vuosilta 2006 ja 2017)

ESCRT-0 ja -1 väliltä

Kaksi artikkelia, joista selviää ESCRT-0 ja -1 komponentteja. Lähinnä kuvataan hiivaa, mutta myös ihmisellä esiintyvistä proteiineista mainitaan. Artikkeli 1 on aika uutta tietoa. Artikkeli vuodelta 2006 antaa tärkeitä perustietoja. Hiivan ESCRT lähinnä fokuksessa, muta ihmisen vastaavia proteiineja mainitaan jos tiedetä

https://ars.els-cdn.com/content/image/1-s2.0-S0962892406001176-gr2.jpg

1. Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo (2017)

https://www.nature.com/articles/oncsis201747

ESCRT-0 ja -1

Abstract

Ubiquitin-dependent sorting of membrane proteins in endosomes directs them to lysosomal degradation. In the case of receptors such as the epidermal growth factor receptor (EGFR), lysosomal degradation is important for the regulation of downstream signalling. Ubiquitinated proteins are recognised in endosomes by the endosomal sorting complexes required for transport (ESCRT) complexes, which sequentially interact with the ubiquitinated cargo. Although the role of each ESCRT complex in sorting is well established, it is not clear how the cargo is passed on from one ESCRT to the next. We here show that flotillin-1 is required for EGFR degradation, and that it interacts with the subunits of ESCRT-0 and -I complexes (hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and Tsg101). Flotillin-1 is required for cargo recognition and sorting by ESCRT-0/Hrs and for its interaction with Tsg101. In addition, flotillin-1 is also required for the sorting of human immunodeficiency virus 1 Gag polyprotein, which mimics ESCRT-0 complex during viral assembly. We propose that flotillin-1 functions in cargo transfer between ESCRT-0 and -I complexes.

Introduction

Signalling by receptors such as the epidermal growth factor receptor (EGFR) is downregulated by degradation of the receptor in lysosomes, which requires the receptor to be ubiquitin modified. Ubiquitinated cargo destined for lysosomal degradation is recognised and sorted by the endosomal sorting complexes required for transport (ESCRTs; reviewed in Henne et al.¹). ESCRTs sequentially bind to the ubiquitinated cargo in endosomes and sequester it into intralumenal vesicles, thereby generating multivesicular bodies that deliver the cargo to lysosomes.^{2,
3} The initial recognition of ubiquitinated cargo and recruitment of ESCRT-I is conducted by the ESCRT-0 complex, which contains two subunits, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and signal-transducing adaptor molecule 1/2 (STAM 1/2). Hrs contains a VHS (Vps27, Hrs and STAM) domain, a lipid-binding FYVE domain,^{4,
5,
6} a UIM (ubiquitin-interacting motif) domain, a coiled-coil domain and a clathrin-binding domain at the C-terminal end.^{7,
8,
9} ESCRT-I is recruited by the binding of its subunit Tsg101 to a P(S/T)AP motif in Hrs, located between the UIM and the coiled-coil domain.^{10,
11,
12,
13} ESCRTs not only have a role during receptor sorting but also many viruses, such as the human immunodeficiency virus 1 (HIV-1), hijack the ESCRT-mediated sorting machinery during particle assembly and viral budding.¹⁴

Flotillin-1 and flotillin-2 are ubiquitously expressed, membrane raft-associated proteins that have been connected to endosomal trafficking of the β-secretase BACE1¹⁵ and of bacterial toxins.^{16,
17} Despite their high degree of conservation, the molecular function of flotillins has remained somewhat obscure. A number of studies have implicated that flotillins may function in membrane trafficking, signalling and cell adhesion by means of their high propensity to oligomerise, and may thus function as scaffolders in various biological processes (for a review, see Meister and Tikkanen¹⁸). However, flotillins can also directly interact with sorting signals in cargo such as BACE1,¹⁵ but their exact role in endosomal trafficking has not been dissected.

Although the role of ESCRTs during degradative sorting of ubiquitinated cargo is well known, the details regarding how the cargo is passed on from one ESCRT to the next during the sorting are not clear. We here show that flotillin-1, which we have previously demonstrated to regulate EGFR signalling but not EGFR uptake from the plasma membrane,¹⁹ is involved in the recognition and sorting of ubiquitinated cargo by ESCRT-0 and -I.

Results

EGFR degradation is attenuated in flotillin-depleted cells

Our earlier findings have shown that although flotillin-1 affects EGFR signalling, it is not involved in the early uptake of EGFR from the plasma membrane upon EGF stimulation.¹⁹ However, upon prolonged EGF stimulation for 60 min, an accumulation of EGFR was observed in intracellular, perinuclear structures in flotillin-1- (Figure 1a) and flotillin-2- (Supplementary Figure S1a) small interfering RNA (siRNA) knockdown HeLa cells, whereas in control siRNA cells, only a very low signal for EGFR was detected (Figure 1a). Quantification showed a significant increase in non-degraded EGFR in flotillin-1-knockdown cells (Figure 1b). Flotillin depletion phenocopied a knockdown of Golgi-localised, γ-ear containing, ADP-ribosylation factor binding protein 3 (GGA3) (Supplementary Figure S1a), which has been shown to impair EGFR degradation.²⁰ Knockdown efficiencies are depicted in Supplementary Figure S1b; please note that flotillin-2 depletion also severely impairs flotillin-1 expression, suggesting that the observed defect is more because of flotillin-1 ablation

än.