Complex compartmentation of tyrosine sulfate-containing proteins undergoing fast axonal transport.
The compartmentation of fast-transported proteins that possess sulfated tyrosine residues--sulfoproteins--has been examined for further resolution of the possible significance of sulfated tyrosine in routing and delivery of fast-transported proteins. In vitro fast axonal transport of [35S]methionine- or 35SO4-labeled proteins was measured in dorsal root ganglion neurons for analysis of protein compartmentation en route and in synaptic regions. When membrane fractions were exposed to Na2CO3 for separation of "lumenal" and peripheral membrane proteins from integral components of the membrane, approximately 20% of the [35S]methionine incorporated into fast-transported proteins was present in a carbonate-releasable form in the axon, whereas 53% of the incorporated 35SO4 was released by carbonate. Eighty percent of the 35SO4 in this releasable fraction was acid labile, typical of sulfate ester-linked to tyrosine. Sulfoproteins were also detected in synaptosomes and were released into the extracellular medium in a calcium-dependent fashion, an observation suggesting that fast-transported sulfoproteins are secreted. Of the remaining 47% of the fast-transported 35SO4-labeled proteins resistant to carbonate treatment (the integral membrane protein fraction), nearly 60% of the 35SO4 was acid labile. Other membrane stripping agents, such as 0.1 M NaOH, 0.5 M NaCl, or mild trypsin treatment, failed to remove acid-labile 35SO4-labeled species from carbonate-treated membrane. Quantitative comparisons of several of the most abundant sulfoproteins resolved via two-dimensional gel electrophoresis confirmed that approximately 7% of each of the species remained associated with carbonate-treated membranes, presumably as integral membrane components.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001370:Axonal Transport; D002254:Carbonates / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005727:Ganglia, Spinal / Q000187:drug effects / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008715:Methionine; D010525:Peripheral Nerves / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011892:Rana catesbeiana; D013116:Spinal Cord / Q000378:metabolism; D013569:Synapses / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D002254:Carbonates; D011506:Proteins; C005662:tyrosine O-sulfate; D014443:Tyrosine; D008715:Methionine