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J. Cell Biol.,
Volume 140, Number 1, January 12, 1998 1-15
* Cell Biology and Metabolism Branch, The ER is uniquely enriched in chaperones
and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and
assembled proteins leave this compartment. Here we
address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are
able to return to the ER folding environment during
their lifetime. Retrieval of proteins back to the ER was
studied using an assay based on the capacity of the ER
to retain misfolded proteins. The lumenal domain of
the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane
targeting domains. At the nonpermissive temperature,
newly synthesized fusion proteins misfolded and were
retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the
ER. At the permissive temperature, the fusion proteins
were correctly delivered to the Golgi complex or
plasma membrane, indicating the lumenal epitope of
VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to
redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were
retained. This occurred over a time period of 15 min-2 h
depending on the chimera, and did not require new
protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within
the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive
VSVGtsO45 lumenal domain to proteins promises to
be a useful tool for studying the molecular mechanisms
and specificity of retrograde traffic to the ER.
National Institute of
Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
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