Profilin I attached to the Golgi is required for the formation of constitutive transport vesicles at the trans-Golgi network.
Profilin I was identified, by mass spectrometric sequencing and immunoblotting, as a component of purified Golgi cisternae from HepG2 cells. Binding to the Golgi was verified by indirect immunofluorescence in MT-1 cells showing that a fraction of profilin I colocalizes with TGN38, a marker of the trans-Golgi network (TGN). Studying the formation of constitutive exocytic vesicles at the TGN in a cell-free system demonstrated that cytosolic profilin I has no effect, while incubation of Golgi cisternae with a profilin I-specific antibody reduced vesicle formation by about 50%. Notably, the antibody displaces a fraction of the Golgi-bound dynamin II indicating that profilin I may indirectly promote vesicle formation by supporting the binding of dynamin II to the Golgi membrane. The impact of dynamin II on vesicle formation is demonstrated by incubating the Golgi with the proline-rich domain of dynamin II which concomitantly displaces dynamin II and inhibits vesicle formation. The data provide evidence that profilin I attaches to the Golgi apparatus and is required for the formation of constitutive transport vesicles.
Dong
J
J
Department of Cell Biology, Max-Delbrück-Centrum for Molecular Medicine, D-13092 Berlin, Germany.
Radau
B
B
Otto
A
A
Müller
E
E
Lindschau
C
C
Westermann
P
P
D003285:Contractile Proteins; D034281:Dynamins; D019084:Fluorescent Antibody Technique, Indirect; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006023:Glycoproteins; D006056:Golgi Apparatus / Q000378:metabolism*; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism*; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000032:analysis; D008565:Membrane Proteins; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism*; D018613:Microscopy, Confocal; D051304:Profilins; D014407:Tumor Cells, Cultured
D003285:Contractile Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D051304:Profilins; D020558:GTP Phosphohydrolases; D034281:Dynamins