A study of the molecular interactions between kinesin motor proteins and the TRAK family of kinesin adaptors
Kinesins are motor proteins that have roles in cell division as well as the transport of various organelles and protein complex cargoes within cells. Kinesin-1 is considered the conventional motor protein and consists of three main isoforms, KIF5A, KIF5B and KIF5C. The family of Trafficking Kinesin...
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ndltd-bl.uk-oai-ethos.bl.uk-6259432015-12-03T03:26:03ZA study of the molecular interactions between kinesin motor proteins and the TRAK family of kinesin adaptorsRandall, T. S.2012Kinesins are motor proteins that have roles in cell division as well as the transport of various organelles and protein complex cargoes within cells. Kinesin-1 is considered the conventional motor protein and consists of three main isoforms, KIF5A, KIF5B and KIF5C. The family of Trafficking Kinesin proteins (TRAKs) bind to the cargo binding domain of kinesin-1 forming a link between the motor protein and their cargoes. The best characterised cargo transported by TRAKs is mitochondria. To understand further the mode of association between kinesin and TRAKs, the aim of this study was to determine the structure of the C-terminal cargo binding domain of kinesin-1. Due to the known binding of TRAK2 with the kinesin-1 family it was hypothesized that TRAKs may stabilize the KIF5A cargo binding domain. Hence, the cDNA encoding the KIF5A cargo binding domain, KIF5A800-1032, and the cDNA encoding the kinesin interacting domain of TRAK2, i.e. TRAK2100-380 were cloned into a bicistronic expression vector to result in epitope-tagged constructs. The expression of both proteins was characterized with respect to yield and solubilisation efficiency. TRAK2100-380 was stabilized by KIF5A800-1032. Affinity chromatography of soluble extracts found that KIF5A800-1032 and TRAK2100-380 did co-purify indicating that they do associate. However, the yield was insufficient for further characterisation. To determine the structure of the cargo binding domain of KIF5A several C-terminal constructs which varied in size and epitope tag location were generated. Bacterial growth and solubilisation conditions were optimized to maximize the yield of the various KIF5A cargo domain recombinant proteins. Each was purified by an appropriate affinity chromatography resin. Conditions were established to optimize the purity, yield, stability and aggregation state. One construct, KIF5A800-951 was isolated to a sufficient yield and did not aggregate. Therefore, KIF5A800-951 is a good candidate for further structural analysis. To refine further the TRAK binding site within the cargo domain of kinesin-1 a series of rationally designed C-terminal truncations of KIF5A and KIF5C were generated and co-expressed with either TRAK1 or TRAK2 in mammalian cells. The binding of kinesin-1 truncations to the TRAKs was determined by co-immunoprecipitation assays followed by quantitative immunoblotting. Deletion of the distal 71 amino acids of KIF5A resulted in an increased co-immunoprecipitation of KIF5A with TRAK2. Three possible TRAK2 binding sites within the KIF5A and KIF5C cargo binding domains were found. Furthermore, differences were found between TRAK1 and TRAK2 in terms of their association sites with KIF5A. Overall these studies yield new insights into kinesin/kinesin adaptor interactions which may impact in the future on a better understanding of neurodegenerative diseases such as hereditary spastic paraplegia and Charcot–Marie–Tooth disease which have both been linked to deficiencies in neuronal transport mechanisms of mitochondria.615.1University College London (University of London)http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.625943http://discovery.ucl.ac.uk/1370581/Electronic Thesis or Dissertation |
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615.1 Randall, T. S. A study of the molecular interactions between kinesin motor proteins and the TRAK family of kinesin adaptors |
description |
Kinesins are motor proteins that have roles in cell division as well as the transport of various organelles and protein complex cargoes within cells. Kinesin-1 is considered the conventional motor protein and consists of three main isoforms, KIF5A, KIF5B and KIF5C. The family of Trafficking Kinesin proteins (TRAKs) bind to the cargo binding domain of kinesin-1 forming a link between the motor protein and their cargoes. The best characterised cargo transported by TRAKs is mitochondria. To understand further the mode of association between kinesin and TRAKs, the aim of this study was to determine the structure of the C-terminal cargo binding domain of kinesin-1. Due to the known binding of TRAK2 with the kinesin-1 family it was hypothesized that TRAKs may stabilize the KIF5A cargo binding domain. Hence, the cDNA encoding the KIF5A cargo binding domain, KIF5A800-1032, and the cDNA encoding the kinesin interacting domain of TRAK2, i.e. TRAK2100-380 were cloned into a bicistronic expression vector to result in epitope-tagged constructs. The expression of both proteins was characterized with respect to yield and solubilisation efficiency. TRAK2100-380 was stabilized by KIF5A800-1032. Affinity chromatography of soluble extracts found that KIF5A800-1032 and TRAK2100-380 did co-purify indicating that they do associate. However, the yield was insufficient for further characterisation. To determine the structure of the cargo binding domain of KIF5A several C-terminal constructs which varied in size and epitope tag location were generated. Bacterial growth and solubilisation conditions were optimized to maximize the yield of the various KIF5A cargo domain recombinant proteins. Each was purified by an appropriate affinity chromatography resin. Conditions were established to optimize the purity, yield, stability and aggregation state. One construct, KIF5A800-951 was isolated to a sufficient yield and did not aggregate. Therefore, KIF5A800-951 is a good candidate for further structural analysis. To refine further the TRAK binding site within the cargo domain of kinesin-1 a series of rationally designed C-terminal truncations of KIF5A and KIF5C were generated and co-expressed with either TRAK1 or TRAK2 in mammalian cells. The binding of kinesin-1 truncations to the TRAKs was determined by co-immunoprecipitation assays followed by quantitative immunoblotting. Deletion of the distal 71 amino acids of KIF5A resulted in an increased co-immunoprecipitation of KIF5A with TRAK2. Three possible TRAK2 binding sites within the KIF5A and KIF5C cargo binding domains were found. Furthermore, differences were found between TRAK1 and TRAK2 in terms of their association sites with KIF5A. Overall these studies yield new insights into kinesin/kinesin adaptor interactions which may impact in the future on a better understanding of neurodegenerative diseases such as hereditary spastic paraplegia and Charcot–Marie–Tooth disease which have both been linked to deficiencies in neuronal transport mechanisms of mitochondria. |
author |
Randall, T. S. |
author_facet |
Randall, T. S. |
author_sort |
Randall, T. S. |
title |
A study of the molecular interactions between kinesin motor proteins and the TRAK family of kinesin adaptors |
title_short |
A study of the molecular interactions between kinesin motor proteins and the TRAK family of kinesin adaptors |
title_full |
A study of the molecular interactions between kinesin motor proteins and the TRAK family of kinesin adaptors |
title_fullStr |
A study of the molecular interactions between kinesin motor proteins and the TRAK family of kinesin adaptors |
title_full_unstemmed |
A study of the molecular interactions between kinesin motor proteins and the TRAK family of kinesin adaptors |
title_sort |
study of the molecular interactions between kinesin motor proteins and the trak family of kinesin adaptors |
publisher |
University College London (University of London) |
publishDate |
2012 |
url |
http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.625943 |
work_keys_str_mv |
AT randallts astudyofthemolecularinteractionsbetweenkinesinmotorproteinsandthetrakfamilyofkinesinadaptors AT randallts studyofthemolecularinteractionsbetweenkinesinmotorproteinsandthetrakfamilyofkinesinadaptors |
_version_ |
1718141371448033280 |