Abstract
We examined the effects of protein folding on endoplasmic reticulum (ER)-to-cytosol transport (dislocation) by exploiting the well-characterized dihydrofolate reductase (DHFR) domain. DHFR retains the capacity to bind folate analogues in the lumen of microsomes and in the ER of intact cells, upon which it acquires a conformation resistant to proteinase K digestion. Here we show that a Class I major histocompatibility complex heavy chain fused to DHFR is still recognized by the human cytomegalovirus-encoded glycoproteins US2 and US11, resulting in dislocation of the fusion protein from the ER in vitro and in vivo. A folded state of the DHFR domain does not impair dislocation of Class I MHC heavy chains in vitro or in living cells. In fact, a slight acceleration of the dislocation of DHFR heavy chain fusion was observed in vitro in the presence of a folate analogue. These results suggest that one or more of the channels used for dislocation can accommodate polypeptides that contain a tightly folded domain of considerable size. Our data raise the possibility that the Sec61 channel can be modified to accommodate a folded DHFR domain for dislocation, but not for translocation into the ER, or that a channel altogether distinct from Sec61 is used for dislocation.
Highlights
The endoplasmic reticulum (ER) to the cytosol, are degraded by the proteasome [3]
The human cytomegalovirus-encoded US2 and US11 glycoproteins mediate the destruction of class I MHC heavy chains (HC) by catalyzing the transfer of Class I HC from the ER to the cytosol, followed by proteasomal degradation, all in a matter of a few minutes
Other studies using dihydrofolate reductase (DHFR) fusion proteins demonstrated that chaperonemediated import into lysosomes, import into chloroplasts, and transport across the bacterial plasma membrane by the Sec machinery require the unfolded conformation for the polypeptide substrate to be translocated, because these processes are all compromised by a folded DHFR domain (10 –12)
Summary
Cell Lines and Antibodies—U373-MG astrocytoma cells (control), and US2 transfectants (US2ϩ) were maintained as described previously [17]. Microsome pellets were resuspended, combined, and lysed together in homogenization buffer containing 1% Triton X-100 with or without proteinase K, as indicated. To measure the extent to which TMX binding to DHFR occurred post-lysis rather than in the ER, the same number of unlabeled U373 cells was exposed to TMX in the same manner These cells were washed twice with cold PBS, and the unlabeled cells were resuspended and combined with pulse-labeled cells. Immunoprecipitates were washed twice with NET buffer (Nonidet P-40 0.5%, NaCl 150 mM, EDTA 5 mM, Tris (pH 7.4) 50 mM), 30 l of homogenization buffer containing 1% Triton X-100 was added, and proteinase K was added to a final concentration of 100 g/ml at room temperature for 45 min.
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