Direct interaction of the trans-Golgi network membrane protein, TGN38, with the F-actin binding protein, neurabin.
TGN38 is a type I integral membrane protein that constitutively cycles between the trans-Golgi network (TGN) and plasma membrane. The cytosolic domain of TGN38 interacts with AP2 clathrin adaptor complexes via the tyrosine-containing motif (-SDYQRL-) to direct internalization from the plasma membrane. This motif has previously been shown to direct both internalization and subsequent TGN targeting of TGN38. We have used the cytosolic domain of TGN38 in a two-hybrid screen, and we have identified the brain-specific F-actin binding protein neurabin-I as a TGN38-binding protein. We demonstrate a direct interaction between TGN38 and the ubiquitous homologue of neurabin-I, neurabin-II (also called spinophilin). We have used a combination of yeast two-hybrid and in vitro protein interaction assays to show that this interaction is dependent on the serine (but not tyrosine) residue of the known TGN38 trafficking motif. We show that TGN38 interacts with the coiled coil region of neurabin in vitro and binds preferentially with the dimeric form of neurabin. TGN38 and neurabin also interact in vivo as demonstrated by coimmunoprecipitation from stably transfected PC12 cells. These data suggest that neurabin provides a direct physical link between TGN38-containing membranes and the actin cytoskeleton.
Stephens
D J
DJ
Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom.
Banting
G
G
D000199:Actins / Q000378:metabolism*; D000818:Animals; D006023:Glycoproteins; D006056:Golgi Apparatus / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins; D008840:Microfilament Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D000099273:Neurabins
D000199:Actins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D000099273:Neurabins; C513045:Tgoln2 protein, rat