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Anterograde trafficking signals in GABAA subunits are required for functional expression.

ABSTRACT: Pentameric GABAA receptors are composed from 19 possible subunits. The GABAA ? subunit is unique because the ?1 and ?3 subunits can assemble and traffic to the cell surface as homomers, whereas most of the other subunits, including ?2, are heteromers. The intracellular domain (ICD) of the GABAA subunits has been implicated in targeting and clustering GABAA receptors at the plasma membrane. Here, we sought to test whether and how the ICD is involved in functional expression of the ?3 subunit. Since ? is the most homologous to ? but does not form homomers, we created two reciprocal chimeric subunits, swapping the ICD between the ?3 and ? subunits, and expressed them in HEK293 cells. Surface expression was detected with immunofluorescence and functional expression was quantified using whole-cell patch-clamp recording with fast perfusion. Results indicate that, unlike ?3, neither the ?3/?IC nor the ?/?3IC chimera can traffic to the plasma membrane when expressed alone; however, when expressed in combination with either wild-type ?3 or ?3, the ?3/?IC chimera was functionally expressed. This suggests that the ICD of ?3 and ?3 each contain essential anterograde trafficking signals that are required to overcome ER retention of assembled GABAA homo- or heteropentamers.

SUBMITTER: Nuwer JL

PROVIDER: S-EPMC6802930 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Anterograde trafficking signals in GABAA subunits are required for functional expression.

Nuwer Jessica L JL Fleck Mark W MW

Channels (Austin, Tex.) 20191201 1

Pentameric GABAA receptors are composed from 19 possible subunits. The GABAA β subunit is unique because the β1 and β3 subunits can assemble and traffic to the cell surface as homomers, whereas most of the other subunits, including β2, are heteromers. The intracellular domain (ICD) of the GABAA subunits has been implicated in targeting and clustering GABAA receptors at the plasma membrane. Here, we sought to test whethe ...[more]

PMID: 31610743

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Project description:UnlabelledThe herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-ΔNLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes.ImportanceThe molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.

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Anterograde trafficking signals in GABAA subunits are required for functional expression.

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Anterograde trafficking signals in GABA<sub>A</sub> subunits are required for functional expression.

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