N‐Linked Glycosylation Is Required for Vacuolar H<sup>+</sup>‐ATPase (V‐ATPase) <i>a4</i> Subunit Stability, Assembly, and Cell Surface Expression
Bibliographic record
Abstract
ABSTRACT The a subunit is the largest of 14 different subunits that make up the V‐ATPase complex. In mammalian species this membrane protein has four paralogous isoforms, a1–a4. Clinically, a subunit isoforms are implicated in diverse diseases; however, little is known about their structure and function. The subunit has conserved, predicted N‐glycosylation sites, and the a3 isoform has been directly shown to be N‐glycosylated. Here we ask if human a4 (ATP6V0A4) is N‐glycosylated at the predicted site, Asn489. We transfected HEK 293 cells, using the pCDNA3.1 expression‐vector system, to express cDNA constructs of epitope‐tagged human a4 subunit, with or without mutations to eliminate the putative glycosylation site. Glycosylation was characterized also by treatment with endoglycosidases; expression and localization were assessed by immunoblotting and immunofluorescence. Endoglycosidase‐treated wild type (WT) a4 showed increased relative mobility on immunoblots, compared with untreated WT a4. This relative mobility was identical to that of unglycosylated mutant a4N489D, demonstrating that the a4 subunit is glycosylated. Cycloheximide pulse‐chase experiments showed that the unglycosylated subunit degraded at a higher rate than the N‐glycosylated form. Unglycosylated a4 was degraded mostly in the proteasomal pathway, but also, in part, through the lysosomal pathway. Immunofluorescence colocalization data showed that unglycosylated a4 was mostly retained in the ER, and that plasma membrane trafficking was defective. Co‐immunoprecipitation studies suggested that a4N489D does not assemble with the V‐ATPase V1 domain. Taken together, these data show that N‐glycosylation plays a crucial role in a4 stability, and in V‐ATPase assembly and trafficking to the plasma membrane. J. Cell. Biochem. 117: 2757–2768, 2016. © 2016 Wiley Periodicals, Inc.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".