Endosomal fusion upon SNARE knockdown is maintained by residual SNARE activity and enhanced docking.
SNARE proteins mediate membrane fusion in the secretory pathway of eukaryotic cells. Genetic deletion and siRNA-based knockdown have been instrumental in assigning given SNAREs to defined intracellular transport steps. However, SNARE depletion occasionally results in barely detectable phenotypes. To understand how cells cope with SNARE loss, we have knocked down several SNAREs functioning in early endosome fusion. Surprisingly, knockdown of syntaxin 13, syntaxin 6 and vti1a, alone or in combinations, did not result in measurable changes of endosomal trafficking or fusion. We found that the residual SNARE levels (typically approximately 10%) were sufficient for a substantial amount of SNARE-SNARE interactions. Conversely, in wild-type cells, most SNARE molecules were concentrated in clusters, constituting a spare pool not readily available for interactions. Additionally, the knockdown organelles exhibited enhanced docking. We conclude that SNAREs are expressed at much higher levels than needed for maintenance of organelle fusion, and that loss of SNAREs is compensated for by the co-regulation of the docking machinery.
Bethani
Ioanna
I
Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Werner
Achim
A
Kadian
Chandini
C
Geumann
Ulf
U
Jahn
Reinhard
R
Rizzoli
Silvio O
SO
D000818:Animals; D015153:Blotting, Western; D002772:Cholera Toxin / Q000378:metabolism; D015536:Down-Regulation; D011992:Endosomes / Q000378:metabolism*; D020868:Gene Silencing; D047468:Immunoprecipitation; D007425:Intracellular Membranes / Q000378:metabolism*; D008561:Membrane Fusion / Q000235:genetics; D008856:Microscopy, Fluorescence; D016716:PC12 Cells; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D050600:SNARE Proteins / Q000235:genetics*; D014162:Transfection
D034741:RNA, Small Interfering; D050600:SNARE Proteins; D002772:Cholera Toxin