Motor proteins are generally considered to move cellular components along microt

Question

Motor proteins are generally considered to move cellular components along microtubules. However, some kinesin family motors may have other functions as described below. a) In one set of experiments, microtubules were observed in a microscope chamber in which the solution could be rapidly exchanged Part (a) of the accompanying figure depicts the effect on microtubule length as a function of lime when the tubulin and buffer in the chamber were replaced first by an identical tubulin and buffer solution and then by buffer alone Part (b) depicts the results when the tubulin and buffer solution in the chamber was replaced with a solution containing tubulin and the M-type kinesin motor protein Kin I. What conclusions can you draw from these experiments? b) In another assay, taxol-stabilized microtubules were incubated alone (Ctrl), with kinesin heavy chain (KMC) and ATP. with Kin I and ATP, or with Kin I and AMPPNP. and then centrifuged to pellet polymerized tubulin Supernatant (s) and pellet (p) fractions were then separated by SDS-PAGE, and the gel was stained to reveal the position of tubulin, as shown in part (c). What conclusions can you draw from these experiments? What is the significance of the nucleotide present for Kin I activity? Would you expect ATP to be present in the experiments depicted in part (b) of the figure? Why or why not? c) Similar results to those shown in part (c) were also obtained when GMPCPP, a nonhydrolyzable GTP analog, was used to assemble stabilized microtubules. What does this result suggest about the action of Kin 1?

Explanation / Answer

Answer:

6. a. Substitution with the tubulin solution has no effect on microtubule growth while replacement with buffer alone causes microtubule shortening because there is no free tubulin to support elongation. In comparison, the plot shown in part (b) indicates that even when free tubulin is available, the Kin I motor acts to promote microtubule shortening (although at a rate slower than results from dilution of the tubulin in the chamber).

b. In the control (Ctrl) and kinesin heavy chain (KHC) samples, all tubulin remained assembled in microtubules as indicated by the presence of tubulin in the pellet fractions. However, Kin I motor in the presence of ATP must have depolymerized the microtubules since the tubulin was found completely in the supernatant fraction (indicating it was no longer assembled).

So, the presence of the nucleotide (ATP) enhances the Kin I activity of depolymerizing the microtubules.

ATP is expected to be present in the experiments depicted in figure (b) since it also shows the microtubule shortening upon addition of Kin I with the buffer.

So, Kin I kinesin can be categorized as a microtubule destabilizing enzyme. The enzyme probably works by targeting directly to microtubule ends where they induce a destabilizing conformational change, facilitated by the hydrolysis of ATP.

c. GMPCPP is a non-hydrolyzable analog of GTP (similar to AMPPNP which is a non-hydrolyzable analog of ATP). Since the results are similar, it can be inferred that Kin I is a microtubule destabilizer and requires the input of energy from the hydrolysis of high energy molecules for its destabilizing activity.

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