Co-localization of cytoskeleton proteins and polysomes with a membrane fraction from peas

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Abstract
When pea (Pisum sativum var. Alaska) stem tissue was ground in the absence of detergent in a cytoskeleton stabilizing buffer and filtrates centrifuged at 300 000×g for 50 min on gradients of 20 to 80% sucrose, a large peak appeared about 2/3 of the way down the gradient. Direct analysis of this peak showed that it contained virtually all of the phospholipids (membranes), about 16% of the total actin and tubulin (cytoskeleton) and about 60% of total tissue polysomes. About 2% of the total cytoskeleton proteins and about 15% of the total polysomes, but no membranes, pelleted to the bottom of the gradient. When a non-ionic detergent was included in the grinding medium, this major peak disappeared, while about 10% of the cytoskeleton proteins, about 65% of the total polysomes, but no membranes, pelleted. When an ionic detergent was included, all of the cytoskeleton proteins were found in the upper regions of the gradient and the polysomes exhibited a normal polysome profile, while no pellet was evident. The addition of 0.2 M KCl in the absence of non-ionic detergent had only a slight effect on the distribution of polysomes and cytoskeleton proteins, indicating that the association is not an artefact caused by a low ionic strength buffer. But in the presence of non-ionic detergent, KCl caused the release of a substantial amount of polysomes, and the cytoskeleton proteins appeared in the uppermost region of the gradient. Based on these results we suggest that: (1) some plant membranes are intimately associated with both the cytoskeleton and with polysomes;(2) membranes help maintain the cohesion of the cytoskeleton; and (3) the cytoskeleton helps maintain the association of polysomes with membranes.

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