Premium
PIKfyve Regulation of Endosome‐Linked Pathways
Author(s) -
de Lartigue Jane,
Polson Hannah,
Feldman Morri,
Shokat Kevan,
Tooze Sharon A.,
Urbé Sylvie,
Clague Michael J.
Publication year - 2009
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/j.1600-0854.2009.00915.x
Subject(s) - endosome , microbiology and biotechnology , biology , endocytic cycle , retromer , gene knockdown , endocytosis , receptor , biochemistry , intracellular , apoptosis
The phosphoinositide 5‐kinase (PIKfyve) is a critical enzyme for the synthesis of PtdIns(3,5) P 2 , that has been implicated in various trafficking events associated with the endocytic pathway. We have now directly compared the effects of siRNA‐mediated knockdown of PIKfyve in HeLa cells with a specific pharmacological inhibitor of enzyme activity. Both approaches induce changes in the distribution of CI‐M6PR and trans‐Golgi network (TGN)‐46 proteins, which cycles between endosomes and TGN, leading to their accumulation in dispersed punctae, whilst the TGN marker golgin‐245 retains a perinuclear disposition. Trafficking of CD8‐CI‐M6PR (retromer‐dependent) and CD8‐Furin (retromer‐independent) chimeras from the cell surface to the TGN is delayed following drug administration, as is the transport of the Shiga toxin B‐subunit. siRNA knockdown of PIKfyve produced no defect in epidermal growth factor receptor (EGFR) degradation, unless combined with knockdown of its activator molecule Vac14, suggesting that a low threshold of PtdIns(3,5) P 2 is necessary and sufficient for this pathway. Accordingly pharmacological inhibition of PIKfyve results in a profound block to the lysosomal degradation of activated epidermal growth factor (EGF) and Met receptors. Immunofluorescence revealed EGF receptors to be trapped in the interior of a swollen endosomal compartment. In cells starved of amino acids, PIKfyve inhibition leads to the accumulation of the lipidated form of GFP‐LC3, a marker of autophagosomal structures, which can be visualized as fluorescent punctae. We suggest that PIKfyve inhibition may render the late endosome/lysosome compartment refractory to fusion with both autophagosomes and with EGFR‐containing multivesicular bodies.