Interaction between p230 and MACF1 is associated with transport of a glycosyl phosphatidyl inositol-anchored protein from the Golgi to the cell periphery.
The molecular basis by which proteins are transported along cytoskeletal tracts from the trans-Golgi network (TGN) to the cell periphery remains poorly understood. Previously, using human autoimmune sera, we identified and characterized a TGN protein, p230/Golgin-245, an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, that is anchored to TGN membranes and TGN-derived vesicles by its carboxyl-terminal GRIP domain. To identify molecules that interact with the flexible amino-terminal end of p230, we used this domain as bait to screen a human brain cDNA library in a yeast two-hybrid assay. We found that this domain interacts with the carboxyl-terminal domain of MACF1, a protein that cross-links microtubules to the actin cytoskeleton. The interaction was confirmed by co-immunoprecipitation, an in vitro binding assay, double immunofluorescence images demonstrating overlapped localization in HeLa cells, and co-localization of FLAG-tagged constructs containing the interacting domains of these two proteins with their endogenous partners. Expression in HeLa cells of FLAG-tagged constructs containing the interacting domains of p230 and MACF1 disrupted transport of the glycosyl phosphatidyl inositol-anchored marker protein conjugated with yellow fluorescent protein (YFP-SP-GPI), while trafficking of the transmembrane marker protein, vesicular stomatitis virus glycoprotein conjugated with YFP (VSVG3-GL-YFP), was unaffected. Our results suggest that p230, through its interaction with MACF1, provides the molecular link for transport of GPI-anchored proteins along the microtubule and actin cytoskeleton from the TGN to the cell periphery.
Kakinuma
Takumi
T
Department of Orthopaedic Surgery, Kyoto University, Kyoto, Japan. t-kak@umin.ac.jp
Ichikawa
Haruo
H
Tsukada
Yoshito
Y
Nakamura
Takashi
T
Toh
Ban-Hock
BH
D000595:Amino Acid Sequence / Q000502:physiology; D001324:Autoantigens / Q000378:metabolism*; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D008870:Microtubules / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001324:Autoantigens; D001426:Bacterial Proteins; C098772:GOLGA4 protein, human; D017261:Glycosylphosphatidylinositols; D008164:Luminescent Proteins; C095003:MACF1 protein, human; D008565:Membrane Proteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria