Sequence and submolecular localization of the 115-kD accessory subunit of the heterotrimeric kinesin-II (KRP85/95) complex.
The heterotrimeric kinesin-II holoenzyme purified from sea urchin (Strongylocentrotus purpuratus) eggs is assembled from two heterodimerized kinesin-related motor subunits of known sequence, together with a third, previously uncharacterized 115-kD subunit, SpKAP115. Using monospecific anti-SpKAP115 antibodies we have accomplished the molecular cloning and sequencing of the SpKAP115 subunit. The deduced sequence predicts a globular 95-kD non-motor "accessory" polypeptide rich in alpha-helical segments that are generally not predicted to form coiled coils. Electron microscopy of individual rotary shadowed kinesin-II holoenzymes also suggests that SpKAP115 is globular, with a somewhat asymmetric morphology. Moreover, the SpKAP115 subunit lies at one end of the 51-nm-long kinesin-II complex, being separated from the two presumptive motor domains by a approximately 26-nm-long rod, in a manner similar to the light chains (KLCs) of kinesin itself. This indicates that SpKAP115 and the KLCs may have analogous functions, yet SpKAP115 does not display significant sequence similarity with the KLCs. The results show that kinesin and kinesin-II are assembled from highly divergent accessory polypeptides together with kinesin related motor subunits (KRPs) containing conserved motor domains linked to divergent tails. Despite the lack of sequence conservation outside the motor domains, there is striking conservation of the ultrastructure of the kinesin and kinesin-II holoenzymes.
Wedaman
K P
KP
Section of Molecular and Cellular Biology, University of California, Davis 95616, USA.
Meyer
D W
DW
Rashid
D J
DJ
Cole
D G
DG
Scholey
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D003001:Cloning, Molecular; D018076:DNA, Complementary; D016547:Kinesins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D009994:Osmolar Concentration; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012617:Sea Urchins
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D046911:Macromolecular Substances; D009124:Muscle Proteins; D011994:Recombinant Proteins; C074152:kinesin-II; D016547:Kinesins