Sequence-Specific Repression of Cotranslational Translocation of the Hepatitis B Virus Envelope Proteins Coincides with Binding of Heat Shock Protein Hsc70

Abstract

The large L envelope protein of the hepatitis B virus has the peculiar capacity to adopt two transmembrane topologies. The N-terminal preS domain of L initially remains in the cytosol while the S domain is cotranslationally inserted into the endoplasmic reticulum membrane. The preS region of about half of the L molecules' is posttranslationally translocated to the lumenal space. We now demonstrate that the repression of cotranslational translocation of preS is conferred by a preS1-specific sequence. By analysis of L deletion mutants, the cytosolic anchorage determinant was mapped to amino acid sequence 70 to 94 of L. The intrinsic potential of this determinant to suppress cotranslational translocation was confirmed by transfer to the HBV middle envelope protein. In searching for cellular factors potentially involved in this novel process, we identified the cytosolic heat shock protein Hsc70 as a specific binding partner of L. The interaction site(s) for the chaperone was mapped to amino acids 63 to 107 of L using coimmunoprecipitation and in vitro binding analyses. Deletion of the cytosolic anchorage determinant almost completely abolished ATP-dependent Hsc70 binding. Therefore, interaction between Hsc70 and L is likely to be responsible for the suppression of cotranslational translocation of the preS domain.

Chaperone action in the posttranslational topological reorientation of the hepatitis B virus large envelope protein: Implications for translocational regulation

“…We demonstrate that L interacts with cytosolic Hsc70 and ER-resident BiP in vivo. Consistent with our previous in vitro results (22), the preS1 domain of L was deemed to be the likely target for Hsc70 and also for BiP because both chaperones failed to recognize the HBV M and S proteins that share the primary sequence of L except for preS1. The lack of chaperone binding of M and S coincides with their classical cotranslational folding pathway and hence provides evidence for functional roles of Hsc70 and BiP in controlling the nonclassical translocation of L. In support, we observed that artificial enforcement of cotranslational preS translocation of L blocked its association with Hsc70, implicating this chaperone to be responsible for the suppression of the cotranslational preS import into the ER.…”

“…2 A, lane 1). N-linked glycosylation of the slower migrating forms had been confirmed previously by treatment with PNGase F (22). Importantly, both forms of L were efficiently coimmunoprecipitated with Hsc70 ( Fig.…”

“…We previously identified Hsc70 as a specific binding partner of the preS1 domain of L in vitro (22) and sought to now determine whether Hsc70 is also associated with the full-length L protein in vivo. COS-7 cells were transiently transfected with an HA-tagged version of the L gene.…”

“…Cytosolic Hsp70s, such as the cognate member Hsc70, in cooperation with Hsp40 class molecules, stimulate the import of proteins into the ER (17,18,20) whereas the luminal Hsp70, the BiP protein (in yeast: Kar2p) is required to provide the driving force for the vectorial movement of polypeptides into the ER lumen (16,19,21). During our search for cellular factors potentially involved in regulating L topogenesis, we previously identified Hsc70 as a specific binding partner of the preS1 domain in vitro (22). Moreover, deletion of the Hsc70-binding site led to cotranslational preS translocation and a uniform luminal topology of L (22), suggesting that L may opt for Hsc70 as a holding and folding device to achieve more than one topological form.…”

“…During our search for cellular factors potentially involved in regulating L topogenesis, we previously identified Hsc70 as a specific binding partner of the preS1 domain in vitro (22). Moreover, deletion of the Hsc70-binding site led to cotranslational preS translocation and a uniform luminal topology of L (22), suggesting that L may opt for Hsc70 as a holding and folding device to achieve more than one topological form. In this study, we examined the putative role of Hsc70 and those of the Hsp40 and BiP chaperones in guiding posttranslational preS translocation.…”