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Abstract Complex N-glycans are asparagine (N)-linked branched sugar chains attached to secretory proteins in eukaryotes. They are produced by modification of N-linked oligosaccharide structures in the endoplasmic reticulum and Golgi apparatus. Complex N-glycans formed in the Golgi apparatus are often assigned specific roles unique to the host organism, with their roles in plants remaining largely unknown. Using inhibitor (kifunensine, KIF) hypersensitivity as read out, we identified Arabidopsis mutants that require complex N-glycan modification. Among >100 KIF-sensitive mutants, one showing abnormal secretory organelles and a salt-sensitive phenotype contained a point mutation leading to amino acid replacement (G69R) in ARFA1E, a small Arf1-GTPase family protein presumably involved in vesicular transport. In vitro assays showed that the G69R exchange interferes with protein activation. In vivo, ARFA1EG69R caused dominant-negative effects, altering the morphology of the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network (TGN). Post-Golgi transport (endocytosis/endocytic recycling) of the essential glycoprotein KORRIGAN1, one of the KIF sensitivity targets, is slowed down constitutively as well as under salt stress in the ARFA1EG69R mutant. Because regulated cycling of plasma membrane proteins is required for stress tolerance of the host plants, the ARFA1EG69R mutant established a link between KIF-targeted luminal glycoprotein functions/dynamics and cytosolic regulators of vesicle transport in endosome-/cell wall-associated tolerance mechanisms.
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The Effects of N-Linked Glycosylation on SLC6 Transporters
Membrane transporters of the solute carrier 6 (SLC6) family mediate various physiological processes by facilitating the translocation of amino acids, neurotransmitters, and other metabolites. In the body, the activity of these transporters is tightly controlled through various post-translational modifications with implications on protein expression, stability, membrane trafficking, and dynamics. While N-linked glycosylation is a universal regulatory mechanism among eukaryotes, a consistent mechanism of how glycosylation affects the SLC6 transporter family remains elusive. It is generally believed that glycans influence transporter stability and membrane trafficking; however, the role of glycosylation on transporter dynamics remains disputable, with differing conclusions among individual transporters across the SLC6 family. In this study, we collected over 1 ms of aggregated all-atom molecular dynamics (MD) simulation data to systematically identify the impact of N-glycans on SLC6 transporter dynamics. We modeled four human SLC6 transporters, the serotonin, dopamine, glycine, and B0AT1 transporters, by first simulating all possible combinations of a glycan attached to each glycosylation site followed by investigating the effect of larger, oligo-N-linked glycans to each transporter. The simulations reveal that glycosylation does not significantly affect the transporter structure but alters the dynamics of the glycosylated extracellular loop and surrounding regions. The structural consequences of glycosylation on the loop dynamics are further emphasized with larger glycan molecules attached. However, no apparent differences in ligand stability or movement of the gating helices were observed, and as such, the simulations suggest that glycosylation does not have a profound effect on conformational dynamics associated with substrate transport.
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- Award ID(s):
- 1845606
- PAR ID:
- 10407592
- Date Published:
- Journal Name:
- Journal of Chemical Information and Modeling
- ISSN:
- 1549-9596
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/acs.jcim.2c00940
