TMEM65

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0005739 mitochondrion	IBA GO_REF:0000033	ACCEPT	Summary: Phylogenetic assignment of TMEM65 to the mitochondrion, matching all focused experimental studies of the protein. Reason: Correct, though the mitochondrial inner membrane (GO:0005743) annotations this gene also carries are more informative about where the protein sits.
GO:0003231 cardiac ventricle development	IBA GO_REF:0000033	KEEP AS NON CORE	Summary: Phylogenetic propagation of a cardiac developmental phenotype seen on Tmem65 knockdown in mouse and zebrafish. Reason: A real organismal phenotype, but distal to the molecular function. It arises from the intercalated-disc/connexin-43 work in zebrafish and mouse, and it would equally be an indirect consequence of impaired mitochondrial calcium handling in cardiomyocytes. Retained as a non-core, downstream process.
GO:1903779 regulation of cardiac conduction	IBA GO_REF:0000033	KEEP AS NON CORE	Summary: Phylogenetic propagation of the conduction phenotype associated with Tmem65 loss. Reason: Downstream organismal process rather than the molecular function. Derived from the connexin-43 mislocalisation phenotype, and confounded by the separate requirement for TMEM65 in mitochondrial calcium efflux in excitable tissue.
GO:0005743 mitochondrial inner membrane	IEA GO_REF:0000044	ACCEPT	Summary: UniProt subcellular-location mapping to the mitochondrial inner membrane. Reason: Matches the direct experimental evidence; this is the established primary location.
GO:0005886 plasma membrane	IEA GO_REF:0000120	KEEP AS NON CORE	Summary: Electronic propagation of a plasma-membrane location from mouse Tmem65. Reason: The plasma-membrane/intercalated-disc pool traces to one cardiac membrane-proteomics study and is not reproduced by any of the mitochondrial studies, which find a cleaved targeting sequence and inner-membrane integration. The two localisations are not formally exclusive, so this is retained rather than removed, but it is not where the characterised function occurs.
GO:0035725 sodium ion transmembrane transport	IEA GO_REF:0000108	UNDECIDED	Summary: Automatically inferred from the contested GO:0005432 calcium:sodium antiporter activity annotation by inter-ontology logical inference. Reason: This annotation is only as sound as the molecular function it is derived from, and that function is the subject of an unresolved dispute that GOA itself records with opposite qualifiers (see the two GO:0005432 entries). If TMEM65 is an obligatory partner of NCLX rather than the transporter, TMEM65 does not itself translocate sodium and this term does not apply. No direct measurement of TMEM65-mediated sodium flux exists.
GO:0005515 protein binding	IPI PMID:32296183 A reference map of the human binary protein interactome.	REMOVE	Summary: Bare protein binding from the HuRI binary interactome screen, covering 37 separate partners. Reason: Uninformative as a molecular function; none of the listed partners corresponds to a characterised TMEM65 complex. No more informative molecular function is supportable from this evidence, so the row is removed rather than rewritten. Removal does not imply the reported interaction is false.
GO:0003231 cardiac ventricle development	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Ensembl Compara projection of the mouse Tmem65 cardiac developmental phenotype. Reason: A real organismal phenotype, but distal to the molecular function. It arises from the intercalated-disc/connexin-43 work in zebrafish and mouse, and it would equally be an indirect consequence of impaired mitochondrial calcium handling in cardiomyocytes. Retained as a non-core, downstream process.
GO:0014704 intercalated disc	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Ensembl Compara projection of the mouse intercalated-disc localisation. Reason: Traces to a single cardiac membrane-proteomics and immunofluorescence study; unreconciled with the inner-membrane localisation established by the mitochondrial literature. Kept because the underlying evidence is experimental and the two pools are not formally exclusive.
GO:0061337 cardiac conduction	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Ensembl Compara projection of the cardiac conduction phenotype. Reason: Organismal process downstream of whichever molecular role is correct; not the gene's core function.
GO:0099093 calcium export from the mitochondrion	IEA GO_REF:0000107	ACCEPT	Summary: Ensembl Compara projection of mouse Tmem65 involvement in mitochondrial calcium export. Reason: This is the one thing all parties to the TMEM65 dispute agree on - loss of TMEM65 abolishes sodium-dependent export of matrix calcium. Core biological process.
GO:1903779 regulation of cardiac conduction	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Ensembl Compara projection of the conduction-regulation phenotype. Reason: Downstream organismal process rather than the molecular function. Derived from the connexin-43 mislocalisation phenotype, and confounded by the separate requirement for TMEM65 in mitochondrial calcium efflux in excitable tissue.
GO:0005886 plasma membrane	IDA GO_REF:0000052	KEEP AS NON CORE	Summary: Human Protein Atlas immunofluorescence assignment to the plasma membrane. Reason: The plasma-membrane/intercalated-disc pool traces to one cardiac membrane-proteomics study and is not reproduced by any of the mitochondrial studies, which find a cleaved targeting sequence and inner-membrane integration. The two localisations are not formally exclusive, so this is retained rather than removed, but it is not where the characterised function occurs.
GO:0005432 calcium:sodium antiporter activity	IDA NOT PMID:40200126 TMEM65 regulates and is required for NCLX-dependent mitochon...	UNDECIDED	Summary: A NOT annotation: Garbincius et al. conclude that TMEM65 is an NCLX binding partner required for sodium-dependent mitochondrial calcium efflux, but is not itself the exchanger, because the TMEM65 effect is abolished by deletion or pharmacological inhibition of NCLX. Reason: GOA carries this GO term on TMEM65 twice with opposite polarity - NOT\|enables IDA from PMID:40200126 and enables IDA from PMID:40691517, both curated by UniProt on the same date. GO has annotated both sides of a live dispute rather than choosing between them, and there is no basis in the published evidence for choosing either. Position B (this annotation) rests on a clean epistatic result: TMEM65 overexpression accelerates matrix calcium efflux in control fibroblasts but has no effect in Nclx-null fibroblasts. Position A rests on reconstitution of purified TMEM65 into liposomes, which a Cell Metab commentary argues cannot distinguish calcium binding from calcium translocation, and on TMEM65-induced exchange in Sf9 mitochondria that lack native activity. No electrophysiological recording of purified TMEM65 exists. UNDECIDED rather than ACCEPT or REMOVE, for both entries; accepting either would assert a resolution the evidence does not support. Supporting Evidence: PMID:40200126 we identify the mitochondrial inner membrane protein TMEM65 as an NCLX binding partner that enhances sodium (Na+)-dependent mCa2+ efflux. Mechanistically, acute pharmacological NCLX inhibition or genetic deletion of NCLX ablates the TMEM65-dependent increase in mCa2+ efflux, and loss-of-function studies show that TMEM65 is required for Na+-dependent mCa2+ efflux. PMID:40200126 These findings suggest that TMEM65 limits net matrix Ca2+ accumulation upon increased cytosolic Ca2+, but that this effect requires NCLX. PMID:41061666 Unfortunately, this experiment cannot distinguish simple binding of Ca2+ to TMEM65 protein embedded in the proteoliposome membrane from actual transport of Ca2+ across the membrane.
GO:0005432 calcium:sodium antiporter activity	IDA PMID:40691517 TMEM65 functions as the mitochondrial Na(+)/Ca(2+) exchanger...	UNDECIDED	Summary: Zhang et al. conclude that TMEM65 is itself the mitochondrial Na+/Ca2+ exchanger, on the basis of exchange activity induced in Sf9 mitochondria that lack native mito-NCX and of purified TMEM65 reconstituted into liposomes. Reason: GOA carries this GO term on TMEM65 twice with opposite polarity - NOT\|enables IDA from PMID:40200126 and enables IDA from PMID:40691517, both curated by UniProt on the same date. GO has annotated both sides of a live dispute rather than choosing between them, and there is no basis in the published evidence for choosing either. Position A (this annotation) has the stronger reductionist design - heterologous reconstitution with NCLX verified absent from the preparation, plus a tissue distribution that tracks mito-NCX activity where NCLX's does not - but its liposome assay measures 45Ca2+ accumulation, which a Cell Metab commentary argues cannot distinguish binding from transport, and no electrophysiology on purified TMEM65 exists. Position B's epistatic result (no TMEM65 effect in Nclx-null cells) is unexplained under Position A. UNDECIDED rather than ACCEPT, for both entries; accepting this one while a NOT annotation of the same term stands would assert a resolution the evidence does not support. Supporting Evidence: PMID:40691517 Heterologous expression of TMEM65 induces Na+/Ca2+ exchange in cells lacking native mito-NCX activity. Moreover, purified, liposome-reconstituted TMEM65 exhibits key mito-NCX features. PMID:40691517 Lastly, we verified that no NCLX was co-purified or co-reconstituted with TMEM65 (Extended Data Fig. 9b–c), ensuring that the observed activity is unrelated to NCLX. PMID:40691517 TMEM65 is highly expressed in the heart and brain but absent in the liver, correlating with mito-NCX activity in these tissues. PMID:41061666 In a recent study published in Nature Cell Biology, Zhang et al. challenge the prevailing view that NCLX is the main mediator of mitochondrial sodium (Na+)/Ca2+ exchange (mito-NCX) by proposing that transmembrane protein 65 (TMEM65) is responsible for this phenomenon.
GO:0005743 mitochondrial inner membrane	IDA PMID:40200126 TMEM65 regulates and is required for NCLX-dependent mitochon...	ACCEPT	Summary: TMEM65 acts at the mitochondrial inner membrane. Reason: Well-established core location, agreed by every study of the protein including both sides of the transport dispute. Supporting Evidence: PMID:40200126 Here we report the identification of TMEM65, an inner mitochondrial membrane protein, as a genetically confirmed regulator of NCLX-dependent mCa2+ efflux and demonstrate its critical role in preventing pathogenic mCa2+ overload in excitable tissues.
GO:0005743 mitochondrial inner membrane	IDA PMID:40691517 TMEM65 functions as the mitochondrial Na(+)/Ca(2+) exchanger...	ACCEPT	Summary: TMEM65 acts at the mitochondrial inner membrane. Reason: Well-established core location, agreed by every study of the protein including both sides of the transport dispute.
GO:0099093 calcium export from the mitochondrion	IDA PMID:40200126 TMEM65 regulates and is required for NCLX-dependent mitochon...	ACCEPT	Summary: Loss of TMEM65 abolishes sodium-dependent export of calcium from the mitochondrial matrix. Reason: This is the one thing all parties to the TMEM65 dispute agree on - the annotation is supported by IDA from both PMID:40200126 and PMID:40691517, which disagree about the mechanism but not about the requirement. Core biological process. Supporting Evidence: PMID:40200126 loss-of-function studies show that TMEM65 is required for Na+-dependent mCa2+ efflux
GO:0099093 calcium export from the mitochondrion	IDA PMID:40691517 TMEM65 functions as the mitochondrial Na(+)/Ca(2+) exchanger...	ACCEPT	Summary: TMEM65 depletion severely impairs mitochondrial sodium/calcium exchange and elevates matrix calcium. Reason: This is the one thing all parties to the TMEM65 dispute agree on - the annotation is supported by IDA from both PMID:40200126 and PMID:40691517, which disagree about the mechanism but not about the requirement. Core biological process. Supporting Evidence: PMID:41061666 Zhang et al. provide compelling evidence supporting a critical role for TMEM65 in mCa2+ extrusion.
GO:0005739 mitochondrion	HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an...	ACCEPT	Summary: High-throughput mitochondrial proteomics places TMEM65 in the mitochondrion. Reason: Correct, though the mitochondrial inner membrane (GO:0005743) annotations this gene also carries are more informative about where the protein sits.
GO:0061337 cardiac conduction	ISS GO_REF:0000024	KEEP AS NON CORE	Summary: Curator-judged orthology transfer of the cardiac conduction phenotype from mouse Tmem65. Reason: Organismal process downstream of whichever molecular role is correct; not the gene's core function.
GO:0005743 mitochondrial inner membrane	IDA PMID:24765583 TMEM65 is a mitochondrial inner-membrane protein.	ACCEPT	Summary: Alkali and digitonin extraction of isolated mitochondria identify TMEM65 as an integral protein of the inner membrane, imported via a cleaved N-terminal targeting sequence. Reason: Founding localisation study; well-supported core location. Supporting Evidence: PMID:24765583 Alkali extraction analysis and digitonin extraction test using isolated mitochondria revealed that TMEM65 is an integral membrane protein that localizes to the inner-membrane of mitochondria.
GO:0005743 mitochondrial inner membrane	IDA PMID:28295037 A mutation in the TMEM65 gene results in mitochondrial myopa...	ACCEPT	Summary: Subcellular fractionation of patient and control fibroblasts confirms inner-membrane residence. Reason: Independent confirmation of the core location, in a study that also links TMEM65 loss to severe mitochondrial disease. Supporting Evidence: PMID:28295037 Subcellular fractionation confirmed that the TMEM65 protein was present in the inner mitochondrial membrane.
GO:0003231 cardiac ventricle development	ISS GO_REF:0000024	KEEP AS NON CORE	Summary: Curator-judged orthology transfer of the cardiac developmental phenotype. Reason: A real organismal phenotype, but distal to the molecular function. It arises from the intercalated-disc/connexin-43 work in zebrafish and mouse, and it would equally be an indirect consequence of impaired mitochondrial calcium handling in cardiomyocytes. Retained as a non-core, downstream process.
GO:0005886 plasma membrane	IDA PMID:26403541 Evolutionarily conserved intercalated disc protein Tmem65 re...	KEEP AS NON CORE	Summary: Cationic silica-bead plasma-membrane enrichment and immunofluorescence identify Tmem65 at the cardiomyocyte surface. Reason: The plasma-membrane/intercalated-disc pool traces to one cardiac membrane-proteomics study and is not reproduced by any of the mitochondrial studies, which find a cleaved targeting sequence and inner-membrane integration. The two localisations are not formally exclusive, so this is retained rather than removed, but it is not where the characterised function occurs. Supporting Evidence: PMID:26403541 We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts.
GO:0014704 intercalated disc	IDA PMID:26403541 Evolutionarily conserved intercalated disc protein Tmem65 re...	KEEP AS NON CORE	Summary: Tmem65 is reported at the cardiac intercalated disc, where it is required for correct localisation of connexin 43. Reason: Traces to a single cardiac membrane-proteomics and immunofluorescence study; unreconciled with the inner-membrane localisation established by the mitochondrial literature. Kept because the underlying evidence is experimental and the two pools are not formally exclusive. Supporting Evidence: PMID:26403541 We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts.
GO:1903779 regulation of cardiac conduction	ISS GO_REF:0000024	KEEP AS NON CORE	Summary: Curator-judged orthology transfer of the conduction-regulation phenotype. Reason: Downstream organismal process rather than the molecular function. Derived from the connexin-43 mislocalisation phenotype, and confounded by the separate requirement for TMEM65 in mitochondrial calcium efflux in excitable tissue.

Gene Description

Existing Annotations Review

Core Functions

References

Suggested Questions for Experts

Suggested Experiments

📚 Additional Documentation

Notes

📄 View Raw YAML