Effect of clathrin assembly lymphoid myeloid leukemia protein depletion on clathrin coat formation.
The endocytic accessory clathrin assembly lymphoid myeloid leukemia protein (CALM) is the ubiquitously expressed homolog of the neuron-specific protein AP180 that has been implicated in the retrieval of synaptic vesicle. Here, we show that CALM associates with the alpha-appendage domain of the AP2 adaptor via the three peptide motifs 420DPF, 375DIF and 489FESVF and to a lesser extent with the amino-terminal domain of the clathrin heavy chain. Reducing clathrin levels by RNA interference did not significantly affect CALM localization, but depletion of AP2 weakens its association with the plasma membrane. In cells, where CALM levels were reduced by RNA interference, AP2 and clathrin remained organized in somewhat enlarged bright fluorescent puncta. Electron microscopy showed that the depletion of CALM drastically affected the clathrin lattice structure. Round-coated buds, which are the predominant features in control cells, were replaced by irregularly shaped buds and long clathrin-coated tubules. Moreover, we noted an increase in the number of very small cages that formed on flat lattices. Furthermore, we noticed a redistribution of endosomal markers and AP1 in cells that were CALM depleted. Taken together, our findings indicate a critical role for CALM in the regulation and orderly progression of coated bud formation at the plasma membrane.
Meyerholz
Anika
A
Department of Cell Biology, Center of Anatomy, Hannover Medical School, Carl-Neuberg Str. 1, D-30625 Hannover, Germany.
Hinrichsen
Lars
L
Groos
Stephanie
S
Esk
Peter-Christopher
PC
Brandes
Gudrun
G
Ungewickell
Ernst J
EJ
D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000096:biosynthesis*; D003034:Coated Pits, Cell-Membrane / Q000648:ultrastructure; D011992:Endosomes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D034041:Monomeric Clathrin Assembly Proteins / Q000172:deficiency / Q000502:physiology*; D034622:RNA Interference; D050656:Transcription Factor AP-2 / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism
D002966:Clathrin; D034041:Monomeric Clathrin Assembly Proteins; C439003:PICALM protein, human; D050656:Transcription Factor AP-2