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Association of the testis-specific TRIM/RBCC protein RNF33/TRIM60 with the cytoplasmic motor proteins KIF3A and KIF3B

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Abstract

The Rnf33/Trim60 gene is temporally transcribed in the preimplantation embryo before being silenced at the blastocyst stage but Rnf33 expression is detected in adult testis of the mouse. The putative RNF33 protein is a tripartite motif (TRIM)/RBCC protein composed of a typical RING zinc finger, a B-box 2, two α-helical coiled-coil segments, and a B30.2 domain. As a first step towards the elucidation of the biologic function of RNF33, we aimed in this study to elucidate proteins that associate with RNF33. RNF33-interacting proteins were first derived by the yeast two-hybrid system followed by co-immunoprecipitation assays. Interacting domains were determined by deletion mapping in genetic and biochemical analyzes. RNF33 was shown to interact with the kinesin-2 family members 3A (KIF3A) and 3B (KIF3B) motor proteins in the heterodimeric form known to transport cargos along the microtubule. Domain mapping showed that the RB and B30.2 domains of RNF33 interacted with the respective carboxyl non-motor domains of KIF3A and KIF3B. Since RNF33 interacted with the carboxyl-terminal tail of the KIF3A–KIF3B heterodimer, the motor head section of KIF3A–KIF3B was free and available for association with designated cargo(s) and movement along the microtubule. Data also suggest that RNF33 most likely interacted with KIF3A–KIF3B independent of the adaptor kinesin-associated protein KAP3. This study is a first demonstration of a TRIM protein, namely RNF33, that interacts with the kinesin molecular motors possibly contributing to kinesin-dependent mobilization of specific cargo(s) along the microtubule in the testis of the mouse.

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Abbreviations

3-AT:

3-amino-triazole

APC:

Adenomatous polyposis coli

COS:

C-terminal subgroup one signature

DTT:

Dithiothreitol

KAP3:

Kinesin superfamily-associated protein 3

KIF3A/KIF3B:

Kinesin-2 family members 3A and 3B

MIS:

Mullerian inhibiting substance

NF-κB:

Nuclear factor-κB

RBCC:

RING-Box-coiled-coil

RFP:

Ret finger protein

RING:

Really interesting new gene, RNF, RING finger protein

SPRY:

SplA and ryanodine receptor

TNFα:

Tumor necrosis factor α

TRIM:

Tripartitate motif

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Acknowledgments

The authors thank Dr. Yu-Chan Chao, Academia Sinica, Taipei, for the baculovirus transfer plasmid and Dr. William O. Hancock, Pennsylvania State University, Pennsylvania for the KIF3A and KIF3B expression plasmids. The authors are also grateful to Min-Chuan Hsu, Wan-Yi Lin, and Yao-Hui Tsai for technical assistance, and Professor Kong-Bung Choo for discussion and editing of the manuscript. This study was supported by the National Science Council (Taiwan) grant NSC-100-2313-B-034-001 to CJH. CCH was supported by the National Science Council (Taiwan) postdoctoral fellowship NSC094-2811-B-075.

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Authors and Affiliations

  1. Department of Animal Science and Institute of Biotechnology, School of Agriculture, Chinese Culture University, 55, Hwa-Kang Road, Yang Ming Shan, Taipei, 111, Taiwan

    Chiu-Jung Huang

  2. Department of Medical Research and Education, Taipei Veterans General Hospital, Shipai, Taipei, 112, Taiwan

    Chih-Cheng Huang & Chih-Chun Chang

  3. Graduate Program, Department of Biotechnology and Laboratory Science in Medicine, School of Biomedical Science and Engineering, National Yang Ming University, Shipai, Taipei, 112, Taiwan

    Chih-Chun Chang

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  1. Chiu-Jung Huang
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Correspondence to Chiu-Jung Huang.

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Huang, CJ., Huang, CC. & Chang, CC. Association of the testis-specific TRIM/RBCC protein RNF33/TRIM60 with the cytoplasmic motor proteins KIF3A and KIF3B. Mol Cell Biochem 360, 121–131 (2012). https://doi.org/10.1007/s11010-011-1050-8

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  • DOI: https://doi.org/10.1007/s11010-011-1050-8

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