Article

Pkh1/2-dependent phosphorylation of Vps27 regulates ESCRT-I recruitment to endosomes

University of Strasbourg, Strasburg, Alsace, France
Molecular biology of the cell (Impact Factor: 4.47). 08/2012; 23(20):4054-64. DOI: 10.1091/mbc.E12-01-0001
Source: PubMed

ABSTRACT

Multivesicular endosomes (MVBs) are major sorting platforms for membrane proteins and participate in plasma membrane protein turnover, vacuolar/lysosomal hydrolase delivery, and surface receptor signal attenuation. MVBs undergo unconventional inward budding, which results in the formation of intraluminal vesicles (ILVs). MVB cargo sorting and ILV formation are achieved by the concerted function of endosomal sorting complex required for transport (ESCRT)-0 to ESCRT-III. The ESCRT-0 subunit Vps27 is a key player in this pathway since it recruits the other complexes to endosomes. Here we show that the Pkh1/Phk2 kinases, two yeast orthologues of the 3-phosphoinositide-dependent kinase, phosphorylate directly Vps27 in vivo and in vitro. We identify the phosphorylation site as the serine 613 and demonstrate that this phosphorylation is required for proper Vps27 function. Indeed, in pkh-ts temperature-sensitive mutant cells and in cells expressing vps27(S613A), MVB sorting of the carboxypeptidase Cps1 and of the α-factor receptor Ste2 is affected and the Vps28-green fluorescent protein ESCRT-I subunit is mainly cytoplasmic. We propose that Vps27 phosphorylation by Pkh1/2 kinases regulates the coordinated cascade of ESCRT complex recruitment at the endosomal membrane.

Download full-text

Full-text

Available from: Joëlle Morvan
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1 delta cells accumulated FM4-64 to a greater extent than wild-type (WT))cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1's implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.
    Full-text · Article · Mar 2013 · Journal of Biosciences
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ent3 and Ent5 are yeast epsin N-terminal homology (ENTH) domain containing proteins involved in protein trafficking between the Golgi and late endosomes (LE). They interact with clathrin, clathrin adaptor at the Golgi (AP-1 and GGA) and different SNAREs (Vti1, Snc1, Pep12 and Syn8) required for vesicular transport at the Golgi and endosomes. To better understand the role of these epsins in membrane trafficking, we performed a protein-protein interaction screen. We identified Btn3/Tda3, a putative oxidoreductase, as a new partner of both Ent3 and Ent5. Btn3 is a negative regulator of the Batten disease linked protein Btn2 involved in the retrieval of specific SNAREs (Vti1, Snc1, Tlg1 and Tlg2) from the LE to the Golgi. We show that Btn3 endosomal localization depends on epsins Ent3 and Ent5. We demonstrated that in btn3Δ mutant cells, endosomal sorting of ubiquitinated cargos and endosomal recycling of the Snc1 SNARE are delayed. We thus propose that Btn3 regulates the sorting function of two adaptors for SNARE proteins, the epsin Ent3 and the Batten disease linked protein Btn2.
    Full-text · Article · Feb 2015 · Journal of Cell Science
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ent3 and Ent5 are yeast epsin N-terminal homology (ENTH) domain containing proteins involved in protein trafficking between the Golgi and late endosomes (LE). They interact with clathrin, clathrin adaptor at the Golgi (AP-1 and GGA) and different SNAREs (Vti1, Snc1, Pep12 and Syn8) required for vesicular transport at the Golgi and endosomes. To better understand the role of these epsins in membrane trafficking, we performed a protein-protein interaction screen. We identified Btn3/Tda3, a putative oxidoreductase, as a new partner of both Ent3 and Ent5. Btn3 is a negative regulator of the Batten disease linked protein Btn2 involved in the retrieval of specific SNAREs (Vti1, Snc1, Tlg1 and Tlg2) from the LE to the Golgi. We show that Btn3 endosomal localization depends on epsins Ent3 and Ent5. We demonstrated that in btn3Δ mutant cells, endosomal sorting of ubiquitinated cargos and endosomal recycling of the Snc1 SNARE are delayed. We thus propose that Btn3 regulates the sorting function of two adaptors for SNARE proteins, the epsin Ent3 and the Batten disease linked protein Btn2.
    Full-text · Article · Feb 2015 · Journal of Cell Science
Show more