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Duke University Medical Center, Department of Cell Biology, Durham, North Carolina 27710
Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and
requires the binding of translationally active ribosomes
to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been
identified in binding studies with inactive ribosomes,
suggesting that ribosome binding is mediated through a
receptor-ligand interaction. To determine if the binding
of nascent chain-bearing ribosomes is regulated in a
manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of
the ER ribosome binding activity, we observed that
Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain
bearing ribosomes to the ER membrane is, however,
insensitive to the ribosome occupancy state of Sec61p.
To determine if additional, Sec61p independent, stages
of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction
was formed, yet the nascent chain was salt extractable
and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were
protease and salt resistant and cross-linked to Sec61p
with higher efficiency. On the basis of these and other
data, we propose that ribosome binding to the ER
membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes
association of the ribosome/nascent chain complex with Sec61p.
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