Differential modes of orphan subunit recognition for the WRB/CAML complex

Inglis, Alison J.; Page, Katharine R.; Guna, Alina; Voorhees, R.M.

doi:10.1101/828228

Cited by 9 publications

(15 citation statements)

References 47 publications

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“…Based on the recent studies showing the translocation of TMD2 of CAML into the ER lumen ( Fig. 6) (25,26), we speculate that the C-TMD (TMD3) of CAML is also retained in the Sec61 translocon in order to assemble with WRB. Alternatively, TMD2 of CAML may be retained in the Sec61 translocon with the C-TMD exposed to the cytosol.…”

Section: Discussionmentioning

confidence: 74%

“…We hypothesized that the slow translocation of C-TMD of WRB is important for efficient assembly with its partner TMD from CAML, which inserts incorrectly in the absence of WRB (25,26) (Fig. 5A).…”

Section: The Sec61 Translocon Complex Serves As a Holdase For Insuffimentioning

confidence: 99%

“…The C-TMD translocation rate determines the assembly efficiency with its partner TMD (A) Left panel: A hypothetic model showing the slow translocation of insufficiently hydrophobic C-TMD of WRB provides a time window for efficient assembly with the partner TMD of CAML. The topology of unassembled CAML is adopted from recent studies (25,26). Right panel: The C-TMD of WRB that either quickly inserts into the membrane or translocates into the ER lumen poorly assembles with the partner TMD of CAML.…”

Section: Quantification and Statistical Analysismentioning

confidence: 99%

“…This retention provides a time window to assemble with CAML, which is likely associated with the Sec61 translocon through its C-TMD or TMD2 (not shown). The topology of unassembled CAML is adopted from recent studies (25,26). If the Sec61 retained TMD fails to be assembled with a partner TMD, it slowly translocates through the translocon into the ER lumen.…”

Section: Quantification and Statistical Analysismentioning

confidence: 99%

“…While this study was under preparation, two recent studies have reported that WRB plays a crucial role in fixing the topology of CAML. In the absence of WRB, CAML exhibits two different topologies: one with both TMD2 and TMD3 translocated into the ER lumen and the other with only TMD2 translocated into the ER lumen (25,26).The molecular mechanism by which newly synthesized WRB and CAML are brought together for efficient assembly is unclear. In this study, we identified that the Sec61 translocon can post-translationally retain membrane proteins.…”

Section: Introductionmentioning

confidence: 99%

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C-terminal tail length guides insertion and assembly of membrane proteins

Sun

Mariappan

2020

Journal of Biological Chemistry

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A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multi-protein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ~60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ~80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins.

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Section: Discussionmentioning

confidence: 74%

Section: The Sec61 Translocon Complex Serves As a Holdase For Insuffimentioning

confidence: 99%

Section: Quantification and Statistical Analysismentioning

confidence: 99%

Section: Quantification and Statistical Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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C-terminal tail length guides insertion and assembly of membrane proteins

Sun

Mariappan

2020

Journal of Biological Chemistry

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WNK1 is an assembly factor for the human ER membrane protein complex

et al. 2021

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The assembly of nascent proteins into multi-subunit complexes is tightly regulated to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble subunits for function. Here we show that the kinase With no lysine 1 (WNK1), known for its role in hypertension and neuropathy, is required for assembly of the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2-8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and precludes binding of ubiquitin ligases, permitting assembly. Using biochemical reconstitution, we show that after EMC2 reaches the membrane, its interaction partners within the EMC displace WNK1, and similarly shield its exposed hydrophobic surfaces. This work describes an unexpected role for WNK1 in protein biogenesis, and defines the general requirements of an assembly factor that will apply across the proteome.

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Cell-type–specific, multicolor labeling of endogenous proteins with split fluorescent protein tags in Drosophila

Kamiyama

Banzai

Liu

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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The impact of the Drosophila experimental system on studies of modern biology cannot be understated. The ability to tag endogenously expressed proteins is essential to maximize the use of this model organism. Here, we describe a method for labeling endogenous proteins with self-complementing split fluorescent proteins (split FPs) in a cell-type–specific manner in Drosophila. A short fragment of an FP coding sequence is inserted into a specific genomic locus while the remainder of the FP is expressed using an available GAL4 driver line. In consequence, complementation fluorescence allows examination of protein localization in particular cells. Besides, when inserting tandem repeats of the short FP fragment at the same genomic locus, we can substantially enhance the fluorescence signal. The enhanced signal is of great value in live-cell imaging at the subcellular level. We can also accomplish a multicolor labeling system with orthogonal split FPs. However, other orthogonal split FPs do not function for in vivo imaging besides split GFP. Through protein engineering and in vivo functional studies, we report a red split FP that we can use for duplexed visualization of endogenous proteins in intricate Drosophila tissues. Using the two orthogonal split FP systems, we have simultaneously imaged proteins that reside in distinct subsynaptic compartments. Our approach allows us to study the proximity between and localization of multiple proteins endogenously expressed in essentially any cell type in Drosophila.

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Differential modes of orphan subunit recognition for the WRB/CAML complex

Abstract: 1

Cited by 9 publications

References 47 publications

C-terminal tail length guides insertion and assembly of membrane proteins

C-terminal tail length guides insertion and assembly of membrane proteins

WNK1 is an assembly factor for the human ER membrane protein complex

Cell-type–specific, multicolor labeling of endogenous proteins with split fluorescent protein tags in Drosophila

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