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Flexibility of the Sec13/31 cage is influenced by the Sec31 C-terminal disordered domain.

ABSTRACT: In COPII mediated vesicle formation, Sec13/Sec31 heterotetramers play a role in organizing the membranes into a spherical vesicle. There they oligomerize into a cage that interacts with the other COPII proteins to direct vesicle formation and concentrate cargo into a bud. In this role they must be flexible to accommodate different sizes and shapes of cargo, but also have elements that provide rigidity to help deform the membrane. Here we characterize the influence the C-terminal disordered region of Sec31 has on cage flexibility and rigidity. After deleting this region (residues 820-1220), we characterized Sec13/Sec31?C heterotetramers biophysically and structurally through cryo-EM. Our results show that Sec13/31?C self-assembles into canonical cuboctahedral cages in vitro at buffer conditions similar to wild type. The distribution of cage sizes indicated that unlike the wild type, Sec13/31?C cages have a more homogeneous geometry. However, the structure of cuboctahedrons exhibited more conformational heterogeneity than wild type. Through localized reconstruction of cage vertices and molecular dynamics flexible fitting we found a new hinge for the flexing of Sec31 ?-propeller domain and more flexibility of the previously known hinge. Together, these results show that the C-terminal region of Sec31 regulates the flexing of other domains such that flexibility and rigidity are not compromised during transport of large and/or asymmetric cargo.

SUBMITTER: Paraan M

PROVIDER: S-EPMC6188663 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Flexibility of the Sec13/31 cage is influenced by the Sec31 C-terminal disordered domain.

Paraan Mohammadreza M Bhattacharya Nilakshee N Uversky Vladimir N VN Stagg Scott M SM

Journal of structural biology 20180830 2

In COPII mediated vesicle formation, Sec13/Sec31 heterotetramers play a role in organizing the membranes into a spherical vesicle. There they oligomerize into a cage that interacts with the other COPII proteins to direct vesicle formation and concentrate cargo into a bud. In this role they must be flexible to accommodate different sizes and shapes of cargo, but also have elements that provide rigidity to help deform the membrane. Here we characterize the influence the C-terminal disordered regio ...[more]

PMID: 30172710

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Project description:Microsporidia, as unicellular eukaryotes, also have an endomembrane system for transporting proteins, which is essentially similar to those of other eukaryotes. In eukaryotes, coat protein complex II (COPII) consists of Sar1, Sec23, Sec24, Sec13, and Sec31 and mediates protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Sar1 is the central player in the regulation of coat protein complex II vesicle formation in the endoplasmic reticulum. In this study, we successfully cloned the NbSar1, NbSec23-1, NbSec23-2, NbSec24-1, NbSec24-2, NbSec13, NbSec31-1, and NbSec31-2 genes and prepared NbSar1 polyclonal antibody. We found that NbSar1 was localized mainly in the perinuclear cytoplasm of Nosema bombycis by immunofluorescence analysis (IFA). Yeast two-hybrid assays demonstrated that NbSar1 interacts with NbSec23-2, NbSec23-2 interacts with NbSec24-1 or NbSec24-2, NbSec23-1 interacts with NbSec31, and NbSec31 interacts with NbSec13. Moreover, the silencing of NbSar1 by RNA interference resulted in the aberrant expression of NbSar1, NbSec23-1, NbSec24-1, NbSec24-2, NbSec13, NbSec31-1, and NbSec31-2 and significantly inhibited the proliferation of N. bombycis. Altogether, these findings indicated that the subunits of coat protein complex II work together to perform functions in the proliferation of N. bombycis and that NbSar1 may play a crucial role in coat protein complex II vesicle formation. IMPORTANCE As eukaryotes, microsporidia have retained the endomembrane system for transporting and sorting proteins throughout their evolution. Whether the microsporidia form coat protein complex II (COPII) vesicles to transport cargo proteins and whether they play other roles besides cargo transport are not fully explained at present. Our results showed that NbSar1, NbSec23-1/NbSec23-2, NbSec24-1/NbSec24-2, NbSec13, and NbSec31 might be assembled to form COPII in the ER of N. bombycis, and the functions of COPII are also closely related to the proliferation of N. bombycis, this may be a new target for the prevention of pébrine disease of the silkworm.

Dataset Information

Flexibility of the Sec13/31 cage is influenced by the Sec31 C-terminal disordered domain.

Publications

Flexibility of the Sec13/31 cage is influenced by the Sec31 C-terminal disordered domain.

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