Inmaculada Navarro-Lérida 1 Mónica Martínez Moreno 1 Fernando Roncal 2 Francisco Gavilanes 1 Juan Pablo Albar 2 Ignacio Rodríguez-Crespo 1 1 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid, Spain 2 Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Madrid, Spain 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/RSTQTor GIQVD motifs in their sequence, hence permitting their transport in a retro- grade 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 dehy- drogenase 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. Keywords: Dynein / Microtubules / Migration / Neuronal proteins / Pepscan PRO 0528 1 Introduction Dyneins are very large multicomponent protein com- plexes that function as microtubule-based molecular motors generating force towards the minus end of micro- tubules [1–3]. They are involved in multiple physiologically relevant cellular processes including mitosis, ciliary/flag- ellar beating and vesicular transport [1–3]. From a struc- tural and functional point of view, the two major classes of dyneins are the axonemal dyneins that intervene in flagel- lar and ciliary movement, and the cytoplasmic dyneins. These transport enzymes contain motor activities, a prop- erty of the two heavy chains (HC; ,530 kDa), each of which forms a multilobed globular head structure where ATP hydrolysis takes place. The HC 1 are associated with copies (probably two) of a 74 kDa intermediate chain (IC), that are thought to function as linkers of cargos to the dynein motor through direct binding to dynactin. In addi- tion, the dynein machinery is formed by four light inter- mediate chains of 50–60 kDa, and several light chains (LCs) with molecular masses lower than 22 kDa. The ICs and LCs are presumably involved in dynein regulatory functions as well as attachment to the appropriate cargo [1–3]. In the case of cytoplasmic dynein, this global asso- ciation of several polypeptide chains results in a total mo- lecular mass of ,1.25 MDa [1–3]. The 8 kDa subunit (LC8, actual mass 10 kDa) was first identified as an integral component of the Chlamydomo- nas outer dynein arm, in association with the ICs at the base of the soluble particle [4]. LC8 protein is a stoi- chiometric component of both brain cytoplasmic dynein and myosin V [5, 6]. Initially, LC8 was cloned and re- ported to be an inhibitor of neuronal nitric oxide synthase (nNOS), capable of inducing the transition of active dimeric nNOS into inactive monomers [7]. Two subse- quent studies [8, 9] were unable to reproduce these results, reinforcing the putative role of LC8 in the trans- port of nNOS along microtubules in the axon rather than abrogating its activity. Since then immunoprecipita- tion, yeast two-hybrid and pepscan studies have identi- fied over twenty cellular proteins that bind to LC8. These include the pro-apoptotic member of the Bcl-2 family Correspondence: Dr. Ignacio Rodríguez-Crespo, Departamento de Bioquímica y Biología Molecular, Faculdad de Ciencias Quí- micas, Universidad Complutense de Madrid, 28040 Madrid, Spain E-mail: nacho@bbml.ucm.es Fax: 134-91-394-4159 Abbreviations: GKAP , guanylate kinase domain-associated pro- tein; HC, heavy chain; LC, light chain; LC8, 8 kDa dynein light chain; Ni, nickel; NMDAR, N-methyl-D-aspartate receptor; nNOS, neuronal nitric oxide synthase; PFK, phosphofructoki- nase; PSD, post-synaptic density; SAPAP , synapse-associated protein 90-associated protein Proteomics 2004, 4, 339–346 339  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de DOI 10.1002/pmic.200300528