SNARE function is not involved in early endosome docking.
Docking and fusion of transport vesicles constitute elementary steps in intracellular membrane traffic. While docking is thought to be initiated by Rab-effector complexes, fusion is mediated by SNARE (N-ethylmaleimide-sensitive factor [NSF] attachment receptor) proteins. However, it has been recently debated whether SNAREs also play a role in the establishment or maintenance of a stably docked state. To address this question, we have investigated the SNARE dependence of docking and fusion of early endosomes, one of the central sorting compartments in the endocytic pathway. A new, fluorescence-based in vitro assay was developed, which allowed us to investigate fusion and docking in parallel. Similar to homotypic fusion, docking of early endosomes is dependent on the presence of ATP and requires physiological temperatures. Unlike fusion, docking is insensitive to the perturbation of SNARE function by means of soluble SNARE motifs, SNARE-specific F(ab) fragments, or by a block of NSF activity. In contrast, as expected, docking is strongly reduced by interfering with the synthesis of phosphatidyl inositol (PI)-3 phosphate, with the function of Rab-GTPases, as well as with early endosomal autoantigen 1 (EEA1), an essential tethering factor. We conclude that docking of early endosomes is independent of SNARE function.
Geumann
Ulf
U
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen 37077, Germany.
Barysch
Sina Victoria
SV
Hoopmann
Peer
P
Jahn
Reinhard
R
Rizzoli
Silvio O
SO
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D016716:PC12 Cells; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins