Proteomic identification of brain proteins that interact with dynein light chain LC8.
Cytoplasmic dynein is a large minus end-directed microtubule motor that translocates cargos towards the minus end of microtubules. Light chain 8 of the dynein machinery (LC8) has been reported to interact with a large variety of proteins that possess K/RSTQT or GIQVD motifs in their sequence, hence permitting their transport in a retrograde manner. Yeast two-hybrid analysis has revealed that in brain, LC8 associates directly with several proteins such as neuronal nitric oxide synthase, guanylate kinase domain-associated protein and gephyrin. In this work, we report the identification of over 40 polypeptides, by means of a proteomic approach, that interact with LC8 either directly or indirectly. Many of the neuronal proteins that we identified cluster at the post-synaptic terminal, and some of them such as phosphofructokinase, lactate dehydrogenase or aldolase are directly involved in glutamate metabolism. Other pool of proteins identified displayed the LC8 consensus binding motif. Finally, recombinant LC8 was produced and a library of overlapping dodecapeptides (pepscan) was employed to map the LC8 binding site of some of the proteins that were previously identified using the proteomic approach, hence confirming binding to the consensus binding sites.
Navarro-Lérida
Inmaculada
I
Departamento de Bioquímicay Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid, Spain. nacho@bbml.ucm.es
Martínez Moreno
Mónica
M
Roncal
Fernando
F
Gavilanes
Francisco
F
Albar
Juan Pablo
JP
Rodríguez-Crespo
Ignacio
I
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D029721:Drosophila Proteins; D004398:Dyneins; D018698:Glutamic Acid / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D011485:Protein Binding; D011506:Proteins / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D000074407:SAP90-PSD95 Associated Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D029721:Drosophila Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D000074407:SAP90-PSD95 Associated Proteins; C073441:gephyrin; D018698:Glutamic Acid; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D004398:Dyneins