KINETICS OF ENTRY OF PROTEINS INTO THE MYELIN MEMBRANE¹

Abstract

Brain slices were prepared from 17‐day old rats, and incubated with [3H]glycine or [3H]‐leucine to label proteins. Myelin was isolated from the slices, and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulfate. Radioactive basic and Wolfgram proteins appeared in myelin at similar initial rates, and their entry was nearly linear between 15 and 120 min with no detectable lag. Radioactive proteolipid protein appeared in myelin at one‐fourth the rate of the basic and Wolfgram proteins between 0 and 30 min, then entered at a rate comparable to the other proteins between 45 and 120 min. When cycloheximide (0.2 mM) or puromycin (1.0 mM) was added, appearance of newly labeled basic and Wolfgram proteins in myelin stopped while proteolipid protein continued to appear in myelin at a normal rate for at least 30 min. Chase experiments with unlabeled glycine had similar effects. These results indicate the existence of a previously synthesized precursor pool of proteolipid protein with a 30‐min interval between synthesis of proteolipid protein and its appearance in myelin. Incorporation of [3H]fucose into glycoprotein of the myelin sheath was studied, as was inhibition of incorporation of radioactivity by the use of either cycloheximide, or dilution with unlabeled fucose. The results indicated fucosylation of a sizable pool of presynthesized protein and a delay of 30 min between fucosylation of these polypeptides and their subsequent appearance in myelin as glycoproteins.

Stabilization of myelin mRNAs as measured in a brain slice system

“…A brain slice system developed by Benjamins et al, (1978) was modified to determine the half-life of myelin mRNAs in the context of actively myelinating oligodendrocytes. Brain slices have been used successfully to study myelin protein synthesis and their incorporation into the myelin sheath (Benjamins et al, 1978;Townsend et al, 1982).…”

Spatial segregation of mRNA encoding myelin-specific proteins.

“…This hypothesis was based on a 20-fold enrichment of MBP mRNA in RNA extracts of myelin fractions when compared to RNA extracts of whole brain homogenates (24). Biochemical studies (24)(25)(26) have shown that MBP enters myelin within a few minutes after synthesis. In contrast, PLP and Po enter myelin about 30 min after synthesis.…”

Functional implications for the microtubule-associated protein tau: localization in oligodendrocytes.

“…It is at the site of myelin assembly that MBP mRNA is translated. To reach its site of translation, this mRNA is translocated long distances from the cell body (20)(21)(22)(23), establishing yet another example of "local synthesis" similar to that occurring at the base of dendritic spines in the neuron (24). Presumably, microtubules are involved in this transport process.…”