Interplay between Disulfide Bonding and N-Glycosylation Defines SLC4 Na+-coupled Transporter Extracellular Topography

Zhu, Qing; Kao, Liyo; Azimov, Rustam; Abuladze, Natalia; Newman, Debra K.; Kurtz, Ira

doi:10.1074/jbc.m114.619320

Cited by 12 publications

(16 citation statements)

References 29 publications

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“…The two disulfide bonds are important for the folded conformation of EL3. Mutations of each of the four highly conserved cysteine residues make EL3 highly accessible to protease digestion 24 . EL3 is also located at the dimeric interface between two subunits and involved in dimerization (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE

et al. 2021

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SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport.

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Section: Resultsmentioning

confidence: 99%

Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE

et al. 2021

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“…As N‐glycosylation and disulfide‐bond formation often occur co‐translationally, and since many proteins comprise a number of both these modifications, it is only natural that these two processes are intricately connected during folding . These connections have been elegantly explored in the Helenius laboratory using a pulse‐chase/immunoprecipitation approach in combination with 2D SDS–PAGE .…”

Section: Interplay Between N‐glycosylation and Disulfide‐bond Formationmentioning

confidence: 99%

Co‐ and Post‐Translational Protein Folding in the ER

Ellgaard

McCaul

Chatsisvili

et al. 2016

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The biophysical rules that govern folding of small, single-domain proteins in dilute solutions are now quite well understood. The mechanisms underlying co-translational folding of multidomain and membrane-spanning proteins in complex cellular environments are often less clear. The endoplasmic reticulum (ER) produces a plethora of membrane and secretory proteins, which must fold and assemble correctly before ER exit -if these processes fail, misfolded species accumulate in the ER or are degraded. The ER differs from other cellular organelles in terms of the physicochemical environment and the variety of ER-specific protein modifications. Here, we review chaperone-assisted co-and post-translational folding and assembly in the ER and underline the influence of protein modifications on these processes. We emphasize how method development has helped advance the field by allowing researchers to monitor the progression of folding as it occurs inside living cells, while at the same time probing the intricate relationship between protein modifications during folding.

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“…It is known that N-linked glycosylation plays a major role in the proper folding and processing of glycoproteins (33,34). Specifically, N-linked glycosylation has been demonstrated to facilitate proper disulfide bond formation (35). In nearly all alphaherpesvirus gKs, there are four extracellular cysteines: three in domain 1 and one in domain 3 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Seminal work of Hammond et al demonstrated a critical role for N-linked glycosylation in viral glycoprotein folding (48), and it has since been shown that the positioning of glycosylation sites is important for optimal protein expression (33). N-linked glycans are critical for directing proper folding of viral and cellular proteins, and proper folding is important for correct disulfide bond formation (35,45,49,50). Not surprisingly, due to the importance of glycosylation to protein processing, mutations in glycosylation have profound effects on virion assembly.…”

Section: Discussionmentioning

confidence: 99%

Cysteines and N-Glycosylation Sites Conserved among All Alphaherpesviruses Regulate Membrane Fusion in Herpes Simplex Virus 1 Infection

Rider

Naderi

Bergeron

et al. 2017

J Virol

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Neurotropism is a defining characteristic of alphaherpesvirus pathogenicity. Glycoprotein K (gK) is a conserved virion glycoprotein of all alphaherpesviruses that is not found in other herpesvirus subfamilies. The extracellular amino terminus of gK has been shown to be important to the ability of the prototypic alphaherpesvirus herpes simplex virus 1 (HSV-1) to enter neurons via axonal termini. Here, we determined the role of the two conserved N-linked glycosylation (N48 and N58) sites of gK in virus-induced cell fusion and replication. We found that N-linked glycosylation is important to the regulation of HSV-1-induced membrane fusion since mutating N58 to alanine (N58A) caused extensive virus-induced cell fusion. Due to the known contributions of N-linked glycosylation to protein processing and correct disulfide bond formation, we investigated whether the conserved extracellular cysteine residues within the amino terminus of gK contributed to the regulation of HSV-1-induced membrane fusion. We found that mutation of C37 and C114 residues led to a gK-null phenotype characterized by very small plaque formation and drastic reduction in infectious virus production, while mutation of C82 and C243 caused extensive virus-induced cell fusion. Comparison of N-linked glycosylation and cysteine mutant replication kinetics identified disparate effects on infectious virion egress from infected cells. Specifically, cysteine mutations caused defects in the accumulation of infectious virus in both the cellular and supernatant fractions, while glycosylation site mutants did not adversely affect virion egress from infected cells. These results demonstrate a critical role for the N glycosylation sites and cysteines for the structure and function of the amino terminus of gK. We have previously identified important entry and neurotropic determinants in the amino terminus of HSV-1 glycoprotein K (gK). Alphaherpesvirus-mediated membrane fusion is a complex and highly regulated process that is not clearly understood. gK and UL20, which are highly conserved across all alphaherpesviruses, play important roles in the regulation of HSV-1 fusion in the context of infection. A greater understanding of mechanisms governing alphaherpesvirus membrane fusion is expected to inform the rational design of therapeutic and prevention strategies to combat herpesviral infection and pathogenesis. This work adds to the growing reports regarding the importance of gK to alphaherpesvirus pathogenesis and details important structural features of gK that are involved in gK-mediated regulation of virus-induced membrane fusion.

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Interplay between Disulfide Bonding and N-Glycosylation Defines SLC4 Na+-coupled Transporter Extracellular Topography

Abstract: Background: Cysteines in extracellular loop 3 (EL-3) are highly conserved in SLC4 Na ϩ -coupled transporters.

Cited by 12 publications

References 29 publications

Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE

Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE

Co‐ and Post‐Translational Protein Folding in the ER

Cysteines and N-Glycosylation Sites Conserved among All Alphaherpesviruses Regulate Membrane Fusion in Herpes Simplex Virus 1 Infection

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