Direct interaction of the 170 kDa isoform of synaptojanin 1 with clathrin and with the clathrin adaptor AP-2.
Synaptojanin 1, a polyphosphoinositide phosphatase, is expressed as two major alternatively spliced isoforms of 145 kDa (SJ145) and 170 kDa (SJ170) [1] [2], which are thought to have pleiotropic roles in endocytosis, signaling and actin function [3] [4] [5]. SJ145 is highly enriched in nerve terminals where it participates in clathrin-dependent synaptic vesicle recycling [1] [5]. SJ170, which differs from SJ145 by the presence of a carboxy-terminal extension, is the predominant isoform in developing neurons and is expressed in a variety of tissues [2]. The carboxy-terminal domain unique to SJ170 was previously shown to bind Eps15 [6], a protein involved in receptor-mediated endocytosis. Here, we show that the same domain also binds clathrin and the clathrin adaptor AP-2. These interactions occur both in vitro and in vivo and are direct. Binding of AP-2 is mediated by the ear domain of its alpha-adaptin subunit and binding of clathrin by the amino-terminal domain of its heavy chain. Overexpression in chinese hamster ovary (CHO) cells of full-length SJ170 or its unique carboxy-terminal region caused mislocalization of Eps15, AP-2 and clathrin, as well as inhibition of clathrin-dependent transferrin uptake. These findings suggest a close association of SJ170 with the clathrin coat and provide new evidence for its physiological role in the regulation of clathrin coat dynamics.
Haffner
C
C
Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA.
Di Paolo
G
G
Rosenthal
J A
JA
de Camilli
P
P
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D016466:CHO Cells; D002966:Clathrin / Q000378:metabolism*; D006224:Cricetinae; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D016716:PC12 Cells; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D014170:Transformation, Genetic
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D007527:Isoenzymes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases