Fast or Slow, Either Head Can Start the Processive Run of Kinesin-2 KIF3AC

Abstract

Mammalian KIF3AC contains two distinct motor polypeptides and is best known for its role in organelle transport in neurons. Our recent studies showed that KIF3AC is as processive as conventional kinesin-1, suggesting that their ATPase mechanochemistry may be similar. However, the presence of two different motor polypeptides in KIF3AC implies that there must be a cellular advantage for the KIF3AC heterodimer. The hypothesis tested was whether there is an intrinsic bias within KIF3AC such that either KIF3A or KIF3C initiates the processive run. To pursue these experiments, a mechanistic approach was used to compare the pre-steady-state kinetics of KIF3AC to the kinetics of homodimeric KIF3AA and KIF3CC. Kinesin-2 subfamily members are best known for their roles in long distance cargo transport, but unlike other processive kinesins, the kinesin-2 subfamily includes both homodimeric and heterodimeric kinesins (reviewed in Refs. 1-3). In mammals, there are four kinesin-2 subfamily genes: KIF3A, KIF3B, KIF3C,. When expressed, KIF17 forms homodimers, yet KIF3A associates with either KIF3B or KIF3C to form the heterodimers KIF3AB and KIF3AC. In each case, these kinesin-2 molecular motors associate with nonmotor adaptor proteins to link the kinesin to its specific cargo (6, 15, 18 -27). Of the kinesin-2 adaptors, the kinesin-associated protein KAP with KIF3AB has resulted in the designation of KIF3AB-KAP as a heterotrimeric kinesin (6, 18 -22, 26). KIF3AB and KIF17 have been widely studied in multiple model organisms including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, as well as Mus musculus to understand their roles in intraflagellar transport and organelle transport for many other cellular functions (1-3). In contrast, KIF3AC has not been as well studied because there is not a true KIF3C ortholog in many of these model organisms. KIF3C exhibits a signature motif conserved in mammalian species, a 25-residue insert in loop L11 of the catalytic motor domain, which is enriched in glycines and serines (7, 28 -30). This L11 insert is not present in other kinesins. Furthermore, even though KIF3AB and KIF3AC share similarities in protein sequence and structure, KIF3AC appears to function specifically in neurons rather than ubiquitously as do KIF3AB and KIF17 (1, 2, 28).Recent single molecule studies revealed that heterodimers of KIF3AC and KIF3AB were as processive as kinesin-1 K560 based on their run lengths, indicating that KIF3AC and KIF3AB have the capacity for long distance intracellular transport (30). Furthermore, to begin to tease apart the contributions of each motor head, homodimers of KIF3AA, KIF3BB, and KIF3CC were generated and characterized. The results for KIF3AA in comparison with KIF3CC were striking because despite sequence similarity between KIF3A and KIF3C, KIF3AA was a very fast and processive kinesin, yet KIF3CC, although processive, was exceedingly slow (Table 1). In contrast, the properties of KIF3AA and KIF3BB were more similar to each other (30,31). This obvious disparity between...

Kinesin-2 heterodimerization alters entry into a processive run along the microtubule but not stepping within the run

“…In contrast, the velocity of KIF3CC was exceedingly slow at 7.5 nm/s with KIF3AC at 186 nm/s, whose faster rate was thought to be due to the properties of KIF3A (35) , both of which are extremely slow (50). Additional evidence that heterodimerization may alter the intrinsic catalytic properties of each motor domain as defined by the engineered homodimers KIF3AA, KIF3BB, and KIF3CC was supported by experimental results that showed that no mixtures of KIF3AA plus KIF3CC or KIF3AA plus KIF3BB could recapitulate the microtubule association and ADP release kinetics of KIF3AC or KIF3AB (49, 50).The general hypothesis has been that heterodimerization provides diversity for adaptor and cargo binding, yet the results to date for KIF3AC and KIF3AB clearly show that in spite of high sequence similarity in their motor domains, KIF3A and KIF3B are both catalytically fast, yet KIF3C is extremely slow (35,(49)(50)(51) To address these questions and gain insight into the motility capability of KIF3AC as a cargo transporter, we repeated the presteady-state kinetics experiments for microtubule association, ADP release, and ATP binding and pursued computational modeling coupled with parameter estimation. The results reveal that the catalytic properties of KIF3A and KIF3C were altered upon microtubule association to be catalytically similar, yet once in the processive run KIF3A remained fast with KIF3C continuing to be slow.…”

“…Additional evidence that heterodimerization may alter the intrinsic catalytic properties of each motor domain as defined by the engineered homodimers KIF3AA, KIF3BB, and KIF3CC was supported by experimental results that showed that no mixtures of KIF3AA plus KIF3CC or KIF3AA plus KIF3BB could recapitulate the microtubule association and ADP release kinetics of KIF3AC or KIF3AB (49,50). puzzling results motivated this study to ask the following questions: 1) How do the properties of KIF3C affect the ATPase properties of KIF3AC?…”

“…Previously, we determined the presteady-state kinetics for microtubule association and native ADP release for KIF3AC, KIF3AA, and KIF3CC (50). For these experiments, the KIF3 motor was rapidly mixed in the stopped-flow instrument with varying concentrations of microtubules plus 50 µM mantATP, a fluorescent analog of ATP.…”

“…Similarly, previous presteady-state kinetics results revealed that microtubule association for KIF3AA and KIF3BB at ~11 µM -1 s -1 coupled with ADP release at ~80 s -1 were both fast, yet microtubule association for KIF3AB was 7 µM (Table 1, (49)). The surprise was that microtubule association for KIF3AC was 6.6 µM -1 s -1 with ADP release at 51 s -1 (50). Yet even though microtubule association by KIF3AC was similar to the constant for KIF3AB, microtubule association for KIF3CC was 2.1 µM -1 s -1 with ADP release at 7.6 s -1…”

“…The general hypothesis has been that heterodimerization provides diversity for adaptor and cargo binding, yet the results to date for KIF3AC and KIF3AB clearly show that in spite of high sequence similarity in their motor domains, KIF3A and KIF3B are both catalytically fast, yet KIF3C is extremely slow (35,(49)(50)(51) To address these questions and gain insight into the motility capability of KIF3AC as a cargo transporter, we repeated the presteady-state kinetics experiments for microtubule association, ADP release, and ATP binding and pursued computational modeling coupled with parameter estimation. The results reveal that the catalytic properties of KIF3A and KIF3C were altered upon microtubule association to be catalytically similar, yet once in the processive run KIF3A remained fast with KIF3C continuing to be slow.…”