Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0005789 endoplasmic reticulum membrane	IBA GO_REF:0000033	ACCEPT	Summary: ROT1 is an ER membrane protein, and this phylogenetic annotation matches direct experimental localization. Reason: ER membrane localization is a core cellular component for Rot1's luminal folding and glycoprotein maturation function. Supporting Evidence: PMID:16567426 Rot1p was found to be an essential, ER-localized membrane protein facing the lumen.
GO:0006458 'de novo' protein folding	IBA GO_REF:0000033	ACCEPT	Summary: Rot1 supports folding and maturation of proteins entering the secretory pathway. Reason: The core function of Rot1 is ER chaperone activity for newly synthesized secretory and membrane proteins. Supporting Evidence: PMID:18508919 Recombinant Rot1 exhibited antiaggregation activity in vitro, which was partly impaired by a temperature-sensitive rot1-2 mutation. PMID:18508919 In vivo, the rot1-2 mutation caused accelerated degradation of five proteins in the secretory pathway via ER-associated degradation, resulting in a decrease in their cellular levels.
GO:0007118 budding cell apical bud growth	IBA GO_REF:0000033	KEEP AS NON CORE	Summary: Bud-growth defects are reproducible consequences of ROT1 inactivation, but they are downstream of ER proteostasis and cell-wall/glycoprotein defects. Reason: Retain as a valid phenotype/process annotation while keeping core function focused on ER folding and glycoprotein maturation. Supporting Evidence: PMID:17606994 First, it is required for the maintenance of apical growth during bud growth.
GO:0051082 unfolded protein binding	IBA GO_REF:0000033	MODIFY	Summary: The evidence supports chaperone activity rather than a generic unfolded protein binding annotation. Reason: GO:0044183 protein folding chaperone better captures Rot1's ER antiaggregation and folding-support activity. Proposed replacements: protein folding chaperone Supporting Evidence: PMID:18508919 Recombinant Rot1 exhibited antiaggregation activity in vitro, which was partly impaired by a temperature-sensitive rot1-2 mutation.
GO:0005783 endoplasmic reticulum	IEA GO_REF:0000002	ACCEPT	Summary: InterPro-derived ER localization is correct but less specific than ER membrane. Reason: Rot1 functions in the ER; the ER membrane term gives the more precise core location. Supporting Evidence: UniProt:Q03691 SUBCELLULAR LOCATION: Endoplasmic reticulum membrane.
GO:0005789 endoplasmic reticulum membrane	IEA GO_REF:0000044	ACCEPT	Summary: UniProtKB subcellular-location mapping to ER membrane is correct. Reason: Rot1 is an ER membrane protein with luminal orientation. Supporting Evidence: UniProt:Q03691 Endoplasmic reticulum membrane; single-pass type I membrane protein.
GO:0006458 'de novo' protein folding	IEA GO_REF:0000002	ACCEPT	Summary: InterPro-based protein-folding annotation is consistent with the primary literature. Reason: Rot1 is an ER chaperone that supports folding/maturation of newly synthesized secretory-pathway proteins. Supporting Evidence: PMID:18508919 Collectively, these results indicate that Rot1 functions as a chaperone in vivo supporting the folding of those proteins. file:yeast/ROT1/ROT1-deep-research-falcon.md In this context, an ER chaperone is a factor that promotes protein folding, prevents aggregation, and/or helps assemble or position protein-processing machinery at the ER translocon.
GO:0005783 endoplasmic reticulum	HDA PMID:26928762 One library to make them all: streamlining the creation of y...	ACCEPT	Summary: High-throughput ER localization is consistent with direct ER-membrane evidence. Reason: ER is the correct compartment for Rot1 function, although ER membrane is more specific. Supporting Evidence: PMID:26928762 Assignments were: Ambiguous; Below threshold; Bud; Bud neck; Cell periphery; Cytosol; ER; Mitochondria; Nuclear periphery; Nucleolus; Nucleus; Punctate; Vacuole; Vacuole membrane.
GO:0005783 endoplasmic reticulum	HDA PMID:14562095 Global analysis of protein localization in budding yeast.	ACCEPT	Summary: High-throughput localization to ER is consistent with direct Rot1 localization studies. Reason: ER localization is core to Rot1 biology.
GO:0006457 protein folding	IGI PMID:16874095 Causal links between protein folding in the ER and events al...	ACCEPT	Summary: Genetic interaction evidence supports Rot1 participation in ER protein folding. Reason: The annotation reflects the core protein-folding role of Rot1 in the ER. Supporting Evidence: PMID:16874095 Rot1 is an essential ER membrane protein and may be involved in protein folding.
GO:0034975 protein folding in endoplasmic reticulum	IGI PMID:16874095 Causal links between protein folding in the ER and events al...	ACCEPT	Summary: This is the best biological-process term for the core Rot1 process. Reason: Rot1 acts in ER protein folding and cooperates with Kar2/BiP. Supporting Evidence: PMID:16567426 Rot1p associated with Kar2p, especially under conditions of ER stress, and maturation of a model protein, a reduced form of carboxypeptidaseY, was impaired in a kar2-1 rot1-2 double mutant.
GO:0005789 endoplasmic reticulum membrane	IDA PMID:16567426 Saccharomyces cerevisiae Rot1p is an ER-localized membrane p...	ACCEPT	Summary: Direct experimental evidence supports ER membrane localization. Reason: Rot1's ER membrane localization is central to its luminal folding and glycosylation-support roles. Supporting Evidence: PMID:16567426 Rot1p was found to be an essential, ER-localized membrane protein facing the lumen.
GO:0006458 'de novo' protein folding	IMP PMID:18508919 Saccharomyces cerevisiae Rot1 is an essential molecular chap...	ACCEPT	Summary: Mutant phenotype evidence directly supports Rot1 as an ER chaperone for newly synthesized secretory-pathway proteins. Reason: This captures Rot1's core ER protein-folding function. Supporting Evidence: PMID:18508919 The rot1-2 mutation caused accelerated degradation of five proteins in the secretory pathway via ER-associated degradation, resulting in a decrease in their cellular levels.
GO:0006458 'de novo' protein folding	IPI PMID:18508919 Saccharomyces cerevisiae Rot1 is an essential molecular chap...	ACCEPT	Summary: Physical interactions with client proteins support Rot1's chaperone role. Reason: The interaction evidence is directly tied to Rot1-dependent folding of secretory-pathway proteins. Supporting Evidence: PMID:18508919 we demonstrate a physical and probably transient interaction of Rot1 with four of these proteins.
GO:0006487 protein N-linked glycosylation	IGI PMID:22492205 The essential endoplasmic reticulum chaperone Rot1 is requir...	ACCEPT	Summary: Rot1 is required for efficient N-glycosylation in sensitized OST backgrounds, especially involving Ost3. Reason: N-glycosylation is a direct core process for Rot1's ER chaperone/assembly-factor function, particularly through the Ost3 branch of the OST machinery. This does not imply Rot1 is itself a catalytic glycosyltransferase subunit. Supporting Evidence: PMID:22492205 OST activity in vitro determined in membranes from rot1-1ost3Delta cells was found to be decreased to 45% compared with wild-type membranes.
GO:0006487 protein N-linked glycosylation	IPI PMID:22492205 The essential endoplasmic reticulum chaperone Rot1 is requir...	ACCEPT	Summary: Physical interaction with Ost3 supports Rot1 involvement in the Ost3-containing OST branch of N-glycosylation. Reason: The Rot1-Ost3 physical interaction supports N-glycosylation as a direct core process coupled to Rot1's ER chaperone/assembly-factor role. Supporting Evidence: PMID:22492205 By affinity chromatography, a physical interaction between Rot1 and Ost3 was demonstrated.
GO:0007118 budding cell apical bud growth	IMP PMID:17606994 Rot1 plays an antagonistic role to Clb2 in actin cytoskeleto...	KEEP AS NON CORE	Summary: Rot1 inactivation disrupts maintenance of apical bud growth. Reason: This is a valid cellular phenotype/process but not the primary molecular function of Rot1. Supporting Evidence: PMID:17606994 Rot1 affects the actin cytoskeleton during the cell cycle at two levels. First, it is required for the maintenance of apical growth during bud growth.
GO:0007118 budding cell apical bud growth	IGI PMID:17606994 Rot1 plays an antagonistic role to Clb2 in actin cytoskeleto...	KEEP AS NON CORE	Summary: Genetic interaction evidence supports the bud-growth phenotype. Reason: The process is downstream of ER proteostasis/cell-wall consequences and is therefore non-core.
GO:0009272 fungal-type cell wall biogenesis	IMP PMID:9545237 Cell wall integrity modulates RHO1 activity via the exchange...	KEEP AS NON CORE	Summary: rot1 mutants have cell-wall defects that affect Rho1/Tor2 pathway phenotypes. Reason: Cell wall biogenesis is a supported downstream process, likely mediated by defective maturation/glycosylation of cell-wall proteins. Supporting Evidence: PMID:9545237 rot1, rot2, big1, cwh41, gas1 and fks1 mutations all confer cell wall defects and suppress tor2(ts).
GO:0030950 establishment or maintenance of actin cytoskeleton polarity	IMP PMID:17606994 Rot1 plays an antagonistic role to Clb2 in actin cytoskeleto...	KEEP AS NON CORE	Summary: Actin-polarity defects are supported by phenotype analysis of rot1 cells. Reason: This is a downstream morphogenesis consequence rather than the core ER molecular function. Supporting Evidence: PMID:17606994 Rot1 is necessary to polarize actin cytoskeleton to the neck region at the end of mitosis.
GO:0035269 protein O-linked glycosylation via mannose	IGI PMID:22492205 The essential endoplasmic reticulum chaperone Rot1 is requir...	KEEP AS NON CORE	Summary: Rot1 perturbation affects O-mannosylation of selected glycoproteins. Reason: This is supported glycosylation biology but is best interpreted as a consequence of Rot1's ER chaperone/assembly role. Supporting Evidence: PMID:22492205 Rot1 was found to be involved also in the O-mannosylation process, as the glycosylation of distinct glycoproteins of this type were affected.
GO:0051082 unfolded protein binding	IDA PMID:18508919 Saccharomyces cerevisiae Rot1 is an essential molecular chap...	MODIFY	Summary: Direct antiaggregation data support chaperone activity, but the binding term is too generic. Reason: Replace with GO:0044183 protein folding chaperone to capture the functional chaperone activity. Proposed replacements: protein folding chaperone Supporting Evidence: PMID:18508919 Recombinant Rot1 exhibited antiaggregation activity in vitro, which was partly impaired by a temperature-sensitive rot1-2 mutation.
GO:0051082 unfolded protein binding	IMP PMID:18508919 Saccharomyces cerevisiae Rot1 is an essential molecular chap...	MODIFY	Summary: Mutant evidence supports impaired protein-folding support, not merely passive unfolded-protein binding. Reason: GO:0044183 is the better functional term for Rot1's chaperone role. Proposed replacements: protein folding chaperone Supporting Evidence: PMID:18508919 Rot1 functions as a chaperone in vivo supporting the folding of those proteins.

Core Functions

Rot1 is an essential ER membrane chaperone or chaperone-associated assembly factor that supports folding and maturation of newly synthesized secretory and membrane proteins. It cooperates with Kar2/BiP and with the Ost3-containing glycosylation machinery, preventing degradation or underglycosylation of a subset of ER clients. N-glycosylation is therefore a direct core process coupled to Rot1-dependent ER proteostasis, while cell-wall, actin-polarity, and bud-growth phenotypes are downstream readouts.

Molecular Function:

protein folding chaperone

Directly Involved In:

protein folding in endoplasmic reticulum 'de novo' protein folding protein N-linked glycosylation

Cellular Locations:

endoplasmic reticulum membrane

Supporting Evidence:

PMID:18508919
Recombinant Rot1 exhibited antiaggregation activity in vitro, which was partly impaired by a temperature-sensitive rot1-2 mutation.
PMID:18508919
Rot1 functions as a chaperone in vivo supporting the folding of those proteins.
file:yeast/ROT1/ROT1-deep-research-falcon.md
Rot1's primary function is best described as an ER chaperone/assembly factor required for efficient glycosylation rather than a catalytic enzyme with a discrete small-molecule substrate.
file:interpro/panther/PTHR28090/PTHR28090-metadata.yaml
PANTHER family PTHR28090 is named PROTEIN ROT1, supporting that the reviewed protein belongs to the conserved Rot1 family.

References

GO_REF:0000002

Gene Ontology annotation through association of InterPro records with GO terms

GO_REF:0000033

Annotation inferences using phylogenetic trees

GO_REF:0000044

Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt

PMID:14562095

Global analysis of protein localization in budding yeast.

PMID:16567426

Saccharomyces cerevisiae Rot1p is an ER-localized membrane protein that may function with BiP/Kar2p in protein folding.

Rot1 is an ER-lumen-facing membrane protein that cooperates with Kar2/BiP.

"Rot1p was found to be an essential, ER-localized membrane protein facing the lumen."

PMID:16874095

Causal links between protein folding in the ER and events along the secretory pathway.

PMID:17606994

Rot1 plays an antagonistic role to Clb2 in actin cytoskeleton dynamics throughout the cell cycle.

PMID:18508919

Saccharomyces cerevisiae Rot1 is an essential molecular chaperone in the endoplasmic reticulum.

Rot1 functions as an ER molecular chaperone.

"Recombinant Rot1 exhibited antiaggregation activity in vitro, and in vivo the rot1-2 mutation caused accelerated degradation of proteins in the secretory pathway."

PMID:22492205

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast.

Rot1 is required for efficient N- and O-glycosylation.

"OST activity in vitro determined in membranes from rot1-1ost3Delta cells was found to be decreased to 45% compared with wild-type membranes."

PMID:26928762

One library to make them all: streamlining the creation of yeast libraries via a SWAp-Tag strategy.

PMID:9545237

Cell wall integrity modulates RHO1 activity via the exchange factor ROM2.

UniProt:Q03691

UniProt entry for ROT1

UniProt summarizes ROT1 as an ER membrane protein involved in protein folding machinery.

"Involved in a protein folding machinery chaperoning proteins acting in various physiological processes including cell wall synthesis."

file:yeast/ROT1/ROT1-deep-research-falcon.md

Falcon deep research report for ROT1

Falcon synthesis supports Rot1 as an ER chaperone/assembly factor linked to glycoprotein maturation.

"Rot1's primary function is best described as an ER chaperone/assembly factor required for efficient glycosylation rather than a catalytic enzyme with a discrete small-molecule substrate."

file:interpro/panther/PTHR28090/PTHR28090-metadata.yaml

PANTHER family PTHR28090 ROT1 metadata

PTHR28090 is a ROT1-specific family supporting conserved Rot1 identity.

"PANTHER family PTHR28090 is named PROTEIN ROT1."

Suggested Experiments

Experiment: Compare ER-associated degradation substrates, glycosylation occupancy, and Rot1 proximity-labeling partners in wild-type, rot1 temperature-sensitive, and complemented strains under permissive and restrictive conditions.

Hypothesis: Rot1 has a restricted client set enriched for secretory-pathway and cell-wall glycoproteins.

Type: client proteomics

Experiment: Reconstitute Ost3-containing and Ost6-containing OST complexes with or without purified Rot1 in microsomal or proteoliposome assays and measure substrate-specific N-glycosylation efficiency.

Hypothesis: Rot1 directly improves Ost3-containing OST function through physical association with Ost3 rather than only indirectly through global ER stress.

Type: glycosylation complex reconstitution

📚 Additional Documentation

Deep Research Falcon

(ROT1-deep-research-falcon.md)

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organism: yeast
gene_id: ROT1
gene_symbol: ROT1
uniprot_accession: Q03691
protein_description: 'RecName: Full=Protein ROT1; AltName: Full=Reversal of TOR2
lethality protein 1; Flags: Precursor;'
gene_info: Name=ROT1; OrderedLocusNames=YMR200W; ORFNames=YM8325.01;
organism_full: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast).
protein_family: Belongs to the ROT1 family. .
protein_domains: Rot1. (IPR019623); Rot1 (PF10681)
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Question

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.

Target Gene/Protein Identity (from UniProt):

UniProt Accession: Q03691
Protein Description: RecName: Full=Protein ROT1; AltName: Full=Reversal of TOR2 lethality protein 1; Flags: Precursor;
Gene Information: Name=ROT1; OrderedLocusNames=YMR200W; ORFNames=YM8325.01;
Organism (full): Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast).
Protein Family: Belongs to the ROT1 family. .
Key Domains: Rot1. (IPR019623); Rot1 (PF10681)

MANDATORY VERIFICATION STEPS:

Check if the gene symbol "ROT1" matches the protein description above
Verify the organism is correct: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast).
Check if protein family/domains align with what you find in literature
If you find literature for a DIFFERENT gene with the same or similar symbol, STOP

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

DO NOT PROCEED WITH RESEARCH ON A DIFFERENT GENE. Instead:
- State clearly: "The gene symbol 'ROT1' is ambiguous or literature is limited for this specific protein"
- Explain what you found (e.g., "Found extensive literature on a different gene with the same symbol in a different organism")
- Describe the protein based ONLY on the UniProt information provided above
- Suggest that the protein function can be inferred from domain/family information

Research Target:

Please provide a comprehensive research report on the gene ROT1 (gene ID: ROT1, UniProt: Q03691) in yeast.

The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.

You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.

We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.

We are interested in where in or outside the cell the gene product carries out its function.

We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.

Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.

Output

Question: You are an expert researcher providing comprehensive, well-cited information.

Provide detailed information focusing on:
1. Key concepts and definitions with current understanding
2. Recent developments and latest research (prioritize 2023-2024 sources)
3. Current applications and real-world implementations
4. Expert opinions and analysis from authoritative sources
5. Relevant statistics and data from recent studies

Format as a comprehensive research report with proper citations. Include URLs and publication dates where available.
Always prioritize recent, authoritative sources and provide specific citations for all major claims.

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.

Target Gene/Protein Identity (from UniProt):

UniProt Accession: Q03691
Protein Description: RecName: Full=Protein ROT1; AltName: Full=Reversal of TOR2 lethality protein 1; Flags: Precursor;
Gene Information: Name=ROT1; OrderedLocusNames=YMR200W; ORFNames=YM8325.01;
Organism (full): Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast).
Protein Family: Belongs to the ROT1 family. .
Key Domains: Rot1. (IPR019623); Rot1 (PF10681)

MANDATORY VERIFICATION STEPS:

Check if the gene symbol "ROT1" matches the protein description above
Verify the organism is correct: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast).
Check if protein family/domains align with what you find in literature
If you find literature for a DIFFERENT gene with the same or similar symbol, STOP

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

Research Target:

Please provide a comprehensive research report on the gene ROT1 (gene ID: ROT1, UniProt: Q03691) in yeast.

The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.

We are interested in where in or outside the cell the gene product carries out its function.

We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.

Research Report: Saccharomyces cerevisiae ROT1 (YMR200W; UniProt Q03691)

Executive summary

ROT1 encodes an essential, endoplasmic reticulum (ER)–localized, multi-pass membrane chaperone (Rot1) whose best-supported primary role is to promote efficient protein glycosylation—especially N-glycosylation via functional coupling to the Ost3-containing oligosaccharyltransferase (OST) complex—and to support maturation/stability of subsets of glycoproteins (including cell-wall–related proteins), with downstream impacts on cell wall integrity and ER proteostasis. (pasikowska2012theessentialendoplasmic pages 1-1, pasikowska2012theessentialendoplasmic pages 1-3)

A key mechanistic anchor is that Rot1 physically associates with Ost3 and that genetic perturbation (rot1-1) combined with loss of OST3 (but not OST6) causes pronounced growth defects, cell-wall drug sensitivities, underglycosylation of multiple N-glycoproteins, and strongly reduced OST activity. (pasikowska2012theessentialendoplasmic pages 1-3, pasikowska2012theessentialendoplasmic media 724ed7b6)

Recent work (2024 preprint) repositions Rot1 as a practical experimental lever: because Rot1 is described as a UPR-independent ER chaperone, inducible Rot1 expression can compensate for loss of canonical UPR signaling (HAC1 deletion) in the context of maintaining euploidy, highlighting Rot1 as a tool to modulate ER folding capacity independent of the UPR transcriptional program. (bartolutti2024uprdeficiencyin pages 5-7)

Identity verification (mandatory)

Target identity (requested): UniProt Q03691, Rot1 family protein “Protein ROT1 / Reversal of TOR2 lethality protein 1,” gene ROT1, systematic locus YMR200W, organism S. cerevisiae S288c.

Verification using retrieved literature: The retrieved primary yeast study uses the gene name ROT1 and protein name Rot1 in S. cerevisiae and describes it as an essential ER chaperone required for N- and O-glycosylation, matching the UniProt description supplied. (pasikowska2012theessentialendoplasmic pages 1-1, pasikowska2012theessentialendoplasmic pages 1-3)

Limitation: The retrieved full texts do not explicitly mention the UniProt accession Q03691; thus, mapping to UniProt is inferred via concordant gene name/protein description and, where available, systematic locus mention in contextual sources. (janik2026theessentialrot1 pages 1-2, pasikowska2012theessentialendoplasmic pages 6-6)

1) Key concepts and definitions (current understanding)

Rot1 as an ER chaperone/assembly factor (definition)

In this context, an ER chaperone is a factor that promotes protein folding, prevents aggregation, and/or helps assemble or position protein-processing machinery at the ER translocon. Rot1 is described as an essential ER-localized protein with chaperone-like/anti-aggregation activity and genetic interaction with the major ER Hsp70 chaperone Kar2/BiP, placing Rot1 in ER folding quality control. (pasikowska2012theessentialendoplasmic pages 1-3, pasikowska2012theessentialendoplasmic pages 6-6)

N-glycosylation via OST and the Ost3/Ost6 isoforms (definition)

N-glycosylation in yeast occurs when the OST complex transfers a preassembled oligosaccharide from a dolichol-linked donor to Asn residues on nascent proteins in the ER. Yeast has OST complexes with alternative subunits Ost3 or Ost6, which can differ in substrate preferences/efficiency. The strongest Rot1-linked phenotypes are specific to the Ost3-containing context (synthetic defects with ost3Δ but not ost6Δ), implicating Rot1 particularly in the Ost3-dependent branch of N-glycosylation. (pasikowska2012theessentialendoplasmic pages 1-3)

O-mannosylation (definition)

Protein O-mannosylation is initiated in the ER by PMT O-mannosyltransferases and often increases on misfolded proteins. Rot1 perturbation affects O-mannosylation for specific substrates and reduces in vitro PMT activity in a sensitized genetic background, linking Rot1 to ER quality control that couples folding state to O-glycan initiation. (pasikowska2012theessentialendoplasmic pages 3-4)

2) Functional annotation: molecular function, processes, and pathways

2.1 Subcellular localization and topology

Rot1 is described as an ER-localized essential membrane protein/chaperone. Its functional effects map to ER luminal processes (OST/PMT activities, folding/quality control), consistent with an ER membrane-embedded protein acting on luminal substrates or luminal domains of glycosylation machinery. (pasikowska2012theessentialendoplasmic pages 8-9, pasikowska2012theessentialendoplasmic pages 6-6)

2.2 Primary molecular function (best-supported)

Rot1’s primary function is best described as an ER chaperone/assembly factor required for efficient glycosylation rather than a catalytic enzyme with a discrete small-molecule substrate. Evidence indicates Rot1 is required for:
- Efficient N-glycosylation (underglycosylation of multiple N-glycoproteins in sensitized mutants; reduced OST activity). (pasikowska2012theessentialendoplasmic pages 1-3, pasikowska2012theessentialendoplasmic pages 3-4)
- O-mannosylation of selected proteins (e.g., Cts1) and reduced in vitro PMT activity under sensitized conditions. (pasikowska2012theessentialendoplasmic pages 3-4, pasikowska2012theessentialendoplasmic pages 6-7)

Mechanistically, Rot1 physically associates with the OST subunit Ost3, supporting a model in which Rot1 helps stabilize/assemble/position the Ost3-containing OST complex at the translocation site for efficient co-translational N-glycosylation. (pasikowska2012theessentialendoplasmic pages 4-5, pasikowska2012theessentialendoplasmic pages 6-6)

2.3 Pathway context

Rot1 sits at the intersection of:
- ER protein folding/proteostasis and UPR-linked stress: rot1 alleles can activate the UPR, consistent with ER folding stress, and Rot1 is genetically connected to Kar2/BiP. (pasikowska2012theessentialendoplasmic pages 6-6)
- N-glycosylation (OST function) and dolichol-linked donor supply: Rot1 perturbation combined with ost3Δ reduces OST activity and is associated with reduced cis-prenyltransferase activity (Rer2-pathway), linking to dolichol precursor synthesis. (pasikowska2012theessentialendoplasmic pages 6-6, pasikowska2012theessentialendoplasmic pages 3-4)
- Cell wall integrity: glycosylation defects of cell-wall proteins and drug sensitivities (e.g., Calcofluor White, Congo Red, caffeine) connect Rot1-dependent glycoprotein maturation to cell wall maintenance. (pasikowska2012theessentialendoplasmic pages 1-1, pasikowska2012theessentialendoplasmic pages 1-3)

3) Key experimental evidence (high-specificity) supporting function

3.1 Genetic interaction specificity with OST3 (not OST6)

In growth/drug assays, rot1-1 alone is reported as only mildly affected under tested conditions, but rot1-1 ost3Δ exhibits abolished growth/temperature sensitivity and enhanced sensitivity to cell wall–perturbing compounds, while rot1-1 ost6Δ does not show the same synthetic interaction—supporting an Ost3-specific functional linkage. (pasikowska2012theessentialendoplasmic pages 1-3)

3.2 In vivo glycosylation readouts (substrate-level)

In the rot1-1 ost3Δ background, multiple model glycoproteins show underglycosylation by immunoblot mobility shifts, including CPY (with forms lacking 1–3 N-glycan chains), DPAPB, Mid2, and Gas1. (pasikowska2012theessentialendoplasmic pages 1-3)

3.3 Enzymatic activity assays: OST and PMT

Biochemical assays on microsomes show:
- Strongly reduced OST activity in rot1-1 ost3Δ relative to wild-type and single mutants. (pasikowska2012theessentialendoplasmic pages 1-3)
- PMT (O-mannosyltransferase) activity reduced to ~45% of wild type in rot1-1 ost3Δ microsomes (quantitative statistic), indicating a measurable O-glycosylation defect under sensitized conditions. (pasikowska2012theessentialendoplasmic pages 3-4)

3.4 Physical interaction between Rot1 and Ost3

Affinity/co-immunoprecipitation experiments show Rot1 and Ost3 co-fractionation consistent with a physical association, supporting a direct mechanistic link between Rot1 and the OST complex. (pasikowska2012theessentialendoplasmic pages 4-5, pasikowska2012theessentialendoplasmic media 724ed7b6)

Figure-based evidence: Cropped figures from the 2012 study document both decreased OST activity in rot1-1 ost3Δ and Rot1–Ost3 co-immunoprecipitation, providing visual support for these key claims. (pasikowska2012theessentialendoplasmic media 724ed7b6, pasikowska2012theessentialendoplasmic media e1a607cd)

4) Recent developments (prioritizing 2023–2024)

4.1 Rot1 as a UPR-independent ER chaperone tool (2024)

A 2024 bioRxiv preprint describes Rot1 explicitly as an ER chaperone not induced by the UPR and uses an aTC-inducible ROT1 allele as a tool to increase ER folding capacity independently of the canonical UPR transcription factor HAC1. Inducing Rot1 expression enabled construction/maintenance of hac1Δ strains (haploid and diploid) while retaining euploidy, and euploidy retention was validated by whole-genome sequencing of 2 independent UPR-deficient populations under induced Rot1. (bartolutti2024uprdeficiencyin pages 5-7)

4.2 Expert framing/interpretation in 2024 work

The 2024 work interprets Rot1 overexpression as evidence that baseline ER folding capacity is a key determinant of whether UPR loss drives adaptive aneuploidy; i.e., increasing chaperone capacity can remove selection for chromosome copy-number changes. This expands Rot1’s role from a glycosylation-linked ER factor to a generalizable experimental handle on ER folding stress outcomes. (bartolutti2024uprdeficiencyin pages 7-9, bartolutti2024uprdeficiencyin pages 5-7)

Note on 2023–2024 peer-reviewed ROT1-focused literature: In the retrieved corpus, direct 2023–2024 peer-reviewed studies specifically centered on S. cerevisiae Rot1 were limited; the principal 2024-specific evidence available here is the above preprint. (bartolutti2024uprdeficiencyin pages 5-7)

5) Current applications and real-world implementations

5.1 Functional genomics / proteostasis engineering

Rot1 is used experimentally in two complementary ways:
1) Mechanistic glycosylation biology: as a sensitizing factor to probe OST isoform function (Ost3 vs Ost6), substrate underglycosylation, and coupling between folding and glycan transfer. (pasikowska2012theessentialendoplasmic pages 1-3, pasikowska2012theessentialendoplasmic pages 3-4)
2) Proteostasis/UPR systems biology: as an inducible, UPR-independent ER chaperone to manipulate ER folding capacity and test causal links between ER stress buffering and genome stability (aneuploidy/euploidy). (bartolutti2024uprdeficiencyin pages 5-7)

5.2 Antifungal-relevant biology (contextual)

Because Rot1 homologs are reported as fungal-restricted in the cited yeast glycosylation work, Rot1-family proteins are of interest as fungal-specific ER proteostasis/glycosylation factors, conceptually aligning with antifungal strategies that target cell wall/glycosylation pathways (though the evidence here is functional/biological rather than a direct drug-development program in S. cerevisiae). (pasikowska2012theessentialendoplasmic pages 7-8, janik2026theessentialrot1 pages 1-2)

6) Relevant statistics and quantitative data (from retrieved studies)

OST activity reduction: In rot1-1 ost3Δ membranes, OST activity is reduced to ~45% of wild type (figure-based evidence). (pasikowska2012theessentialendoplasmic media 724ed7b6)
PMT activity reduction: PMT activity in rot1-1 ost3Δ microsomes is ~45% of wild type (quantitative enzymatic statistic). (pasikowska2012theessentialendoplasmic pages 3-4)
Growth/drug assay conditions (quantitative implementation details): Spot assays used serial 10-fold dilutions starting at 3 × 10^4 cells, with media containing 5 µg/mL Calcofluor White, 5 mM caffeine, 15 µg/mL Congo Red, or 20 µg/mL hygromycin B, incubated 72 h (drug tests at 25°C; temperature sensitivity at 37°C). (pasikowska2012theessentialendoplasmic pages 1-3)
Replicates: Enzymatic activity data are described as averages of three independent experiments. (pasikowska2012theessentialendoplasmic pages 3-4)
2024 population counts (genome stability application): Euploidy retention with induced Rot1 was validated by WGS in 2 independent populations; for comparison, inducible KAR2 prevented aneuploidy in six independent populations. (bartolutti2024uprdeficiencyin pages 5-7)

Evidence map (quick-reference)

The table below links major annotation claims to their strongest supporting sources, including publication dates and URLs.

Aspect	Findings	Best supporting citation IDs (pqac-...)	Publication (authors, year)	URL	Publication date/month if available
identity	ROT1 in this context corresponds to the essential yeast ER protein Rot1 from Saccharomyces cerevisiae; the literature cited describes the same fungal ER chaperone linked to glycosylation and cell-wall integrity, consistent with UniProt Q03691 / YMR200W.	(janik2026theessentialrot1 pages 1-2, pasikowska2012theessentialendoplasmic pages 1-3)	Janik et al., 2026; Pasikowska et al., 2012	https://doi.org/10.3390/jof12040244 ; https://doi.org/10.1093/glycob/cws068	Mar 2026; Apr 2012
localization/topology	Rot1 is an essential ER-localized membrane protein/chaperone; available evidence supports ER membrane residence with an ER-luminal functional role and reported multi-pass topology.	(pasikowska2012theessentialendoplasmic pages 8-9, pasikowska2012theessentialendoplasmic pages 6-6)	Pasikowska et al., 2012	https://doi.org/10.1093/glycob/cws068	Apr 2012
molecular function	Current best-supported function is not an enzyme with a defined catalytic substrate, but an ER chaperone/assembly factor required for efficient protein N-glycosylation and also affecting O-mannosylation, likely by supporting glycosylation machinery and folding of specific substrates.	(pasikowska2012theessentialendoplasmic pages 1-1, pasikowska2012theessentialendoplasmic pages 1-3, pasikowska2012theessentialendoplasmic pages 3-4)	Pasikowska et al., 2012	https://doi.org/10.1093/glycob/cws068	Apr 2012
pathway/process	Rot1 functions in ER protein-folding quality control, N-glycosylation via the Ost3-containing OST complex, O-mannosylation, dolichol-linked glycosylation precursor biology, and downstream cell-wall biogenesis/integrity.	(pasikowska2012theessentialendoplasmic pages 1-3, pasikowska2012theessentialendoplasmic pages 3-4)	Pasikowska et al., 2012	https://doi.org/10.1093/glycob/cws068	Apr 2012
key experimental evidence	Evidence includes genetic interaction with OST3 and KAR2, physical interaction with Ost3 by co-immunoprecipitation/affinity purification, glycosylation defects in model glycoproteins (e.g., CPY, Gas1, Mid2, Cts1, Kre9), and cell-wall stress phenotypes in mutant backgrounds.	(pasikowska2012theessentialendoplasmic pages 1-1, pasikowska2012theessentialendoplasmic pages 4-5, pasikowska2012theessentialendoplasmic media 724ed7b6)	Pasikowska et al., 2012	https://doi.org/10.1093/glycob/cws068	Apr 2012
quantitative/statistics	In rot1-1 ost3Δ membranes, OST activity was reduced to about 45% of wild type; in vitro Pmt/O-mannosyltransferase activity was also reported at about 45% of wild type, providing quantitative support for dual glycosylation defects.	(pasikowska2012theessentialendoplasmic pages 3-4, pasikowska2012theessentialendoplasmic media 724ed7b6)	Pasikowska et al., 2012	https://doi.org/10.1093/glycob/cws068	Apr 2012
recent developments 2024	A 2024 preprint used aTC-inducible ROT1 expression in budding yeast and reported that Rot1, described as a UPR-independent ER chaperone, could support viability of hac1Δ cells and help maintain euploidy, highlighting Rot1 as a tool to probe ER folding capacity independent of canonical UPR signaling.	(bartolutti2024uprdeficiencyin pages 5-7, bartolutti2024uprdeficiencyin pages 7-9, bartolutti2024uprdeficiencyin pages 9-11)	Bartolutti et al., 2024	https://doi.org/10.1101/2024.11.22.624941	Nov 2024
applications/implementations	Rot1 is used experimentally as a genetic and cell-biology handle to study ER proteostasis, glycosylation efficiency, UPR compensation, and fungal cell-wall integrity; because Rot1-family proteins are fungal-specific in the cited literature, they are also discussed as potential antifungal-relevant biology rather than a direct current therapeutic target in S. cerevisiae.	(janik2026theessentialrot1 pages 1-2, bartolutti2024uprdeficiencyin pages 5-7, bartolutti2024uprdeficiencyin pages 7-9)	Janik et al., 2026; Bartolutti et al., 2024	https://doi.org/10.3390/jof12040244 ; https://doi.org/10.1101/2024.11.22.624941	Mar 2026; Nov 2024

Table: This table summarizes the main validated findings for Saccharomyces cerevisiae ROT1/YMR200W, including identity, localization, function, pathway context, quantitative evidence, and recent 2024 developments. It is useful as a compact evidence map linking each claim to the strongest available citation contexts.

Conclusions for functional annotation

1) Molecular function: Rot1 is best annotated as an essential ER membrane chaperone/assembly factor that supports efficient glycoprotein maturation, with strong evidence for a role in Ost3-dependent N-glycosylation and measurable effects on O-mannosylation for selected substrates. (pasikowska2012theessentialendoplasmic pages 1-3, pasikowska2012theessentialendoplasmic pages 3-4)
2) Localization: Endoplasmic reticulum membrane, acting on luminal folding/glycosylation processes. (pasikowska2012theessentialendoplasmic pages 8-9, pasikowska2012theessentialendoplasmic pages 6-6)
3) Pathways/processes: ER proteostasis/quality control, N-glycosylation (OST), selected O-mannosylation (PMT), and downstream cell wall integrity. (pasikowska2012theessentialendoplasmic pages 1-1, pasikowska2012theessentialendoplasmic pages 3-4)
4) Recent 2024 development: Rot1 can be deployed as a UPR-independent ER chaperone tool to experimentally decouple ER folding capacity from canonical UPR signaling and interrogate genome stability outcomes. (bartolutti2024uprdeficiencyin pages 5-7)

Key references (retrieved in this analysis)

Pasikowska M, Palamarczyk G, Lehle L. “The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast.” Glycobiology (Apr 2012). https://doi.org/10.1093/glycob/cws068 (pasikowska2012theessentialendoplasmic pages 1-1)
Bartolutti C, Kim AJ, Brar GA. “UPR deficiency in budding yeast reveals a trade-off between ER folding capacity and maintenance of euploidy.” bioRxiv (Nov 2024). https://doi.org/10.1101/2024.11.22.624941 (bartolutti2024uprdeficiencyin pages 5-7)

References

(pasikowska2012theessentialendoplasmic pages 1-1): M. Pasikowska, G. Palamarczyk, and L. Lehle. The essential endoplasmic reticulum chaperone rot1 is required for protein n- and o-glycosylation in yeast. Glycobiology, 22:939-947, Apr 2012. URL: https://doi.org/10.1093/glycob/cws068, doi:10.1093/glycob/cws068. This article has 11 citations and is from a peer-reviewed journal.
(pasikowska2012theessentialendoplasmic pages 1-3): M. Pasikowska, G. Palamarczyk, and L. Lehle. The essential endoplasmic reticulum chaperone rot1 is required for protein n- and o-glycosylation in yeast. Glycobiology, 22:939-947, Apr 2012. URL: https://doi.org/10.1093/glycob/cws068, doi:10.1093/glycob/cws068. This article has 11 citations and is from a peer-reviewed journal.
(pasikowska2012theessentialendoplasmic media 724ed7b6): M. Pasikowska, G. Palamarczyk, and L. Lehle. The essential endoplasmic reticulum chaperone rot1 is required for protein n- and o-glycosylation in yeast. Glycobiology, 22:939-947, Apr 2012. URL: https://doi.org/10.1093/glycob/cws068, doi:10.1093/glycob/cws068. This article has 11 citations and is from a peer-reviewed journal.
(bartolutti2024uprdeficiencyin pages 5-7): Constantine Bartolutti, Allison J Kim, and Gloria A Brar. Upr deficiency in budding yeast reveals a trade-off between er folding capacity and maintenance of euploidy. bioRxiv, Nov 2024. URL: https://doi.org/10.1101/2024.11.22.624941, doi:10.1101/2024.11.22.624941. This article has 1 citations.
(janik2026theessentialrot1 pages 1-2): Anna Janik, Ewa Zatorska, Urszula Perlińska-Lenart, Sebastian Piłsyk, and Joanna S. Kruszewska. The essential rot1 protein links glycosylation, cell wall integrity, and pathogenic development in candida albicans. Journal of Fungi, 12:244, Mar 2026. URL: https://doi.org/10.3390/jof12040244, doi:10.3390/jof12040244. This article has 0 citations.
(pasikowska2012theessentialendoplasmic pages 6-6): M. Pasikowska, G. Palamarczyk, and L. Lehle. The essential endoplasmic reticulum chaperone rot1 is required for protein n- and o-glycosylation in yeast. Glycobiology, 22:939-947, Apr 2012. URL: https://doi.org/10.1093/glycob/cws068, doi:10.1093/glycob/cws068. This article has 11 citations and is from a peer-reviewed journal.
(pasikowska2012theessentialendoplasmic pages 3-4): M. Pasikowska, G. Palamarczyk, and L. Lehle. The essential endoplasmic reticulum chaperone rot1 is required for protein n- and o-glycosylation in yeast. Glycobiology, 22:939-947, Apr 2012. URL: https://doi.org/10.1093/glycob/cws068, doi:10.1093/glycob/cws068. This article has 11 citations and is from a peer-reviewed journal.
(pasikowska2012theessentialendoplasmic pages 8-9): M. Pasikowska, G. Palamarczyk, and L. Lehle. The essential endoplasmic reticulum chaperone rot1 is required for protein n- and o-glycosylation in yeast. Glycobiology, 22:939-947, Apr 2012. URL: https://doi.org/10.1093/glycob/cws068, doi:10.1093/glycob/cws068. This article has 11 citations and is from a peer-reviewed journal.
(pasikowska2012theessentialendoplasmic pages 6-7): M. Pasikowska, G. Palamarczyk, and L. Lehle. The essential endoplasmic reticulum chaperone rot1 is required for protein n- and o-glycosylation in yeast. Glycobiology, 22:939-947, Apr 2012. URL: https://doi.org/10.1093/glycob/cws068, doi:10.1093/glycob/cws068. This article has 11 citations and is from a peer-reviewed journal.
(pasikowska2012theessentialendoplasmic pages 4-5): M. Pasikowska, G. Palamarczyk, and L. Lehle. The essential endoplasmic reticulum chaperone rot1 is required for protein n- and o-glycosylation in yeast. Glycobiology, 22:939-947, Apr 2012. URL: https://doi.org/10.1093/glycob/cws068, doi:10.1093/glycob/cws068. This article has 11 citations and is from a peer-reviewed journal.
(pasikowska2012theessentialendoplasmic media e1a607cd): M. Pasikowska, G. Palamarczyk, and L. Lehle. The essential endoplasmic reticulum chaperone rot1 is required for protein n- and o-glycosylation in yeast. Glycobiology, 22:939-947, Apr 2012. URL: https://doi.org/10.1093/glycob/cws068, doi:10.1093/glycob/cws068. This article has 11 citations and is from a peer-reviewed journal.
(bartolutti2024uprdeficiencyin pages 7-9): Constantine Bartolutti, Allison J Kim, and Gloria A Brar. Upr deficiency in budding yeast reveals a trade-off between er folding capacity and maintenance of euploidy. bioRxiv, Nov 2024. URL: https://doi.org/10.1101/2024.11.22.624941, doi:10.1101/2024.11.22.624941. This article has 1 citations.
(pasikowska2012theessentialendoplasmic pages 7-8): M. Pasikowska, G. Palamarczyk, and L. Lehle. The essential endoplasmic reticulum chaperone rot1 is required for protein n- and o-glycosylation in yeast. Glycobiology, 22:939-947, Apr 2012. URL: https://doi.org/10.1093/glycob/cws068, doi:10.1093/glycob/cws068. This article has 11 citations and is from a peer-reviewed journal.
(bartolutti2024uprdeficiencyin pages 9-11): Constantine Bartolutti, Allison J Kim, and Gloria A Brar. Upr deficiency in budding yeast reveals a trade-off between er folding capacity and maintenance of euploidy. bioRxiv, Nov 2024. URL: https://doi.org/10.1101/2024.11.22.624941, doi:10.1101/2024.11.22.624941. This article has 1 citations.

Citations

bartolutti2024uprdeficiencyin pages 5-7
pasikowska2012theessentialendoplasmic pages 1-3
pasikowska2012theessentialendoplasmic pages 3-4
pasikowska2012theessentialendoplasmic pages 6-6
pasikowska2012theessentialendoplasmic pages 1-1
pasikowska2012theessentialendoplasmic pages 8-9
pasikowska2012theessentialendoplasmic pages 6-7
pasikowska2012theessentialendoplasmic pages 4-5
bartolutti2024uprdeficiencyin pages 7-9
pasikowska2012theessentialendoplasmic pages 7-8
bartolutti2024uprdeficiencyin pages 9-11
https://doi.org/10.3390/jof12040244
https://doi.org/10.1093/glycob/cws068
https://doi.org/10.1101/2024.11.22.624941
https://doi.org/10.1093/glycob/cws068,
https://doi.org/10.1101/2024.11.22.624941,
https://doi.org/10.3390/jof12040244,

📄 View Raw YAML

id: Q03691
gene_symbol: ROT1
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:559292
  label: Saccharomyces cerevisiae
description: >-
  ROT1 encodes an essential endoplasmic-reticulum membrane protein that acts as
  an ER protein-folding chaperone or chaperone-associated assembly factor. Rot1
  faces the ER lumen, genetically and physically cooperates with ER folding and
  glycosylation factors such as Kar2/BiP and Ost3, prevents aggregation in vitro,
  and supports maturation of secretory and membrane proteins. Its defects cause
  impaired ER protein folding, reduced N- and O-glycosylation of glycoproteins,
  cell wall defects, altered actin polarity, and bud-growth phenotypes; these
  downstream phenotypes are best interpreted as consequences of the core ER
  proteostasis/glycoprotein maturation role.
existing_annotations:
- term:
    id: GO:0005789
    label: endoplasmic reticulum membrane
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      ROT1 is an ER membrane protein, and this phylogenetic annotation matches
      direct experimental localization.
    action: ACCEPT
    reason: >-
      ER membrane localization is a core cellular component for Rot1's luminal
      folding and glycoprotein maturation function.
    supported_by:
    - reference_id: PMID:16567426
      supporting_text: >-
        Rot1p was found to be an essential, ER-localized membrane protein facing
        the lumen.
- term:
    id: GO:0006458
    label: '''de novo'' protein folding'
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      Rot1 supports folding and maturation of proteins entering the secretory
      pathway.
    action: ACCEPT
    reason: >-
      The core function of Rot1 is ER chaperone activity for newly synthesized
      secretory and membrane proteins.
    supported_by:
    - reference_id: PMID:18508919
      supporting_text: >-
        Recombinant Rot1 exhibited antiaggregation activity in vitro, which was
        partly impaired by a temperature-sensitive rot1-2 mutation.
    - reference_id: PMID:18508919
      supporting_text: >-
        In vivo, the rot1-2 mutation caused accelerated degradation of five
        proteins in the secretory pathway via ER-associated degradation,
        resulting in a decrease in their cellular levels.
- term:
    id: GO:0007118
    label: budding cell apical bud growth
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      Bud-growth defects are reproducible consequences of ROT1 inactivation, but
      they are downstream of ER proteostasis and cell-wall/glycoprotein defects.
    action: KEEP_AS_NON_CORE
    reason: >-
      Retain as a valid phenotype/process annotation while keeping core function
      focused on ER folding and glycoprotein maturation.
    supported_by:
    - reference_id: PMID:17606994
      supporting_text: >-
        First, it is required for the maintenance of apical growth during bud
        growth.
- term:
    id: GO:0051082
    label: unfolded protein binding
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      The evidence supports chaperone activity rather than a generic unfolded
      protein binding annotation.
    action: MODIFY
    reason: >-
      GO:0044183 protein folding chaperone better captures Rot1's ER
      antiaggregation and folding-support activity.
    proposed_replacement_terms:
    - id: GO:0044183
      label: protein folding chaperone
    supported_by:
    - reference_id: PMID:18508919
      supporting_text: >-
        Recombinant Rot1 exhibited antiaggregation activity in vitro, which was
        partly impaired by a temperature-sensitive rot1-2 mutation.
- term:
    id: GO:0005783
    label: endoplasmic reticulum
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: >-
      InterPro-derived ER localization is correct but less specific than ER
      membrane.
    action: ACCEPT
    reason: >-
      Rot1 functions in the ER; the ER membrane term gives the more precise core
      location.
    supported_by:
    - reference_id: UniProt:Q03691
      supporting_text: 'SUBCELLULAR LOCATION: Endoplasmic reticulum membrane.'
- term:
    id: GO:0005789
    label: endoplasmic reticulum membrane
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  review:
    summary: >-
      UniProtKB subcellular-location mapping to ER membrane is correct.
    action: ACCEPT
    reason: >-
      Rot1 is an ER membrane protein with luminal orientation.
    supported_by:
    - reference_id: UniProt:Q03691
      supporting_text: >-
        Endoplasmic reticulum membrane; single-pass type I membrane protein.
- term:
    id: GO:0006458
    label: '''de novo'' protein folding'
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: >-
      InterPro-based protein-folding annotation is consistent with the primary
      literature.
    action: ACCEPT
    reason: >-
      Rot1 is an ER chaperone that supports folding/maturation of newly
      synthesized secretory-pathway proteins.
    supported_by:
    - reference_id: PMID:18508919
      supporting_text: >-
        Collectively, these results indicate that Rot1 functions as a chaperone
        in vivo supporting the folding of those proteins.
    - reference_id: file:yeast/ROT1/ROT1-deep-research-falcon.md
      supporting_text: >-
        In this context, an **ER chaperone** is a factor that promotes protein
        folding, prevents aggregation, and/or helps assemble or position
        protein-processing machinery at the ER translocon.
- term:
    id: GO:0005783
    label: endoplasmic reticulum
  evidence_type: HDA
  original_reference_id: PMID:26928762
  review:
    summary: >-
      High-throughput ER localization is consistent with direct ER-membrane
      evidence.
    action: ACCEPT
    reason: >-
      ER is the correct compartment for Rot1 function, although ER membrane is
      more specific.
    supported_by:
    - reference_id: PMID:26928762
      supporting_text: >-
        Assignments were: Ambiguous; Below threshold; Bud; Bud neck; Cell
        periphery; Cytosol; ER; Mitochondria; Nuclear periphery; Nucleolus;
        Nucleus; Punctate; Vacuole; Vacuole membrane.
- term:
    id: GO:0005783
    label: endoplasmic reticulum
  evidence_type: HDA
  original_reference_id: PMID:14562095
  review:
    summary: >-
      High-throughput localization to ER is consistent with direct Rot1
      localization studies.
    action: ACCEPT
    reason: >-
      ER localization is core to Rot1 biology.
- term:
    id: GO:0006457
    label: protein folding
  evidence_type: IGI
  original_reference_id: PMID:16874095
  review:
    summary: >-
      Genetic interaction evidence supports Rot1 participation in ER protein
      folding.
    action: ACCEPT
    reason: >-
      The annotation reflects the core protein-folding role of Rot1 in the ER.
    supported_by:
    - reference_id: PMID:16874095
      supporting_text: >-
        Rot1 is an essential ER membrane protein and may be involved in protein
        folding.
- term:
    id: GO:0034975
    label: protein folding in endoplasmic reticulum
  evidence_type: IGI
  original_reference_id: PMID:16874095
  review:
    summary: >-
      This is the best biological-process term for the core Rot1 process.
    action: ACCEPT
    reason: >-
      Rot1 acts in ER protein folding and cooperates with Kar2/BiP.
    supported_by:
    - reference_id: PMID:16567426
      supporting_text: >-
        Rot1p associated with Kar2p, especially under conditions of ER stress,
        and maturation of a model protein, a reduced form of carboxypeptidaseY,
        was impaired in a kar2-1 rot1-2 double mutant.
- term:
    id: GO:0005789
    label: endoplasmic reticulum membrane
  evidence_type: IDA
  original_reference_id: PMID:16567426
  review:
    summary: >-
      Direct experimental evidence supports ER membrane localization.
    action: ACCEPT
    reason: >-
      Rot1's ER membrane localization is central to its luminal folding and
      glycosylation-support roles.
    supported_by:
    - reference_id: PMID:16567426
      supporting_text: >-
        Rot1p was found to be an essential, ER-localized membrane protein facing
        the lumen.
- term:
    id: GO:0006458
    label: '''de novo'' protein folding'
  evidence_type: IMP
  original_reference_id: PMID:18508919
  review:
    summary: >-
      Mutant phenotype evidence directly supports Rot1 as an ER chaperone for
      newly synthesized secretory-pathway proteins.
    action: ACCEPT
    reason: >-
      This captures Rot1's core ER protein-folding function.
    supported_by:
    - reference_id: PMID:18508919
      supporting_text: >-
        The rot1-2 mutation caused accelerated degradation of five proteins in
        the secretory pathway via ER-associated degradation, resulting in a
        decrease in their cellular levels.
- term:
    id: GO:0006458
    label: '''de novo'' protein folding'
  evidence_type: IPI
  original_reference_id: PMID:18508919
  review:
    summary: >-
      Physical interactions with client proteins support Rot1's chaperone role.
    action: ACCEPT
    reason: >-
      The interaction evidence is directly tied to Rot1-dependent folding of
      secretory-pathway proteins.
    supported_by:
    - reference_id: PMID:18508919
      supporting_text: >-
        we demonstrate a physical and probably transient interaction of Rot1
        with four of these proteins.
- term:
    id: GO:0006487
    label: protein N-linked glycosylation
  evidence_type: IGI
  original_reference_id: PMID:22492205
  review:
    summary: >-
      Rot1 is required for efficient N-glycosylation in sensitized OST
      backgrounds, especially involving Ost3.
    action: ACCEPT
    reason: >-
      N-glycosylation is a direct core process for Rot1's ER
      chaperone/assembly-factor function, particularly through the Ost3 branch
      of the OST machinery. This does not imply Rot1 is itself a catalytic
      glycosyltransferase subunit.
    supported_by:
    - reference_id: PMID:22492205
      supporting_text: >-
        OST activity in vitro determined in membranes from rot1-1ost3Delta cells
        was found to be decreased to 45% compared with wild-type membranes.
- term:
    id: GO:0006487
    label: protein N-linked glycosylation
  evidence_type: IPI
  original_reference_id: PMID:22492205
  review:
    summary: >-
      Physical interaction with Ost3 supports Rot1 involvement in the
      Ost3-containing OST branch of N-glycosylation.
    action: ACCEPT
    reason: >-
      The Rot1-Ost3 physical interaction supports N-glycosylation as a direct
      core process coupled to Rot1's ER chaperone/assembly-factor role.
    supported_by:
    - reference_id: PMID:22492205
      supporting_text: By affinity chromatography, a physical interaction between Rot1 and Ost3 was demonstrated.
- term:
    id: GO:0007118
    label: budding cell apical bud growth
  evidence_type: IMP
  original_reference_id: PMID:17606994
  review:
    summary: >-
      Rot1 inactivation disrupts maintenance of apical bud growth.
    action: KEEP_AS_NON_CORE
    reason: >-
      This is a valid cellular phenotype/process but not the primary molecular
      function of Rot1.
    supported_by:
    - reference_id: PMID:17606994
      supporting_text: >-
        Rot1 affects the actin cytoskeleton during the cell cycle at two levels.
        First, it is required for the maintenance of apical growth during bud
        growth.
- term:
    id: GO:0007118
    label: budding cell apical bud growth
  evidence_type: IGI
  original_reference_id: PMID:17606994
  review:
    summary: >-
      Genetic interaction evidence supports the bud-growth phenotype.
    action: KEEP_AS_NON_CORE
    reason: >-
      The process is downstream of ER proteostasis/cell-wall consequences and is
      therefore non-core.
- term:
    id: GO:0009272
    label: fungal-type cell wall biogenesis
  evidence_type: IMP
  original_reference_id: PMID:9545237
  review:
    summary: >-
      rot1 mutants have cell-wall defects that affect Rho1/Tor2 pathway
      phenotypes.
    action: KEEP_AS_NON_CORE
    reason: >-
      Cell wall biogenesis is a supported downstream process, likely mediated by
      defective maturation/glycosylation of cell-wall proteins.
    supported_by:
    - reference_id: PMID:9545237
      supporting_text: >-
        rot1, rot2, big1, cwh41, gas1 and fks1 mutations all confer cell wall
        defects and suppress tor2(ts).
- term:
    id: GO:0030950
    label: establishment or maintenance of actin cytoskeleton polarity
  evidence_type: IMP
  original_reference_id: PMID:17606994
  review:
    summary: >-
      Actin-polarity defects are supported by phenotype analysis of rot1 cells.
    action: KEEP_AS_NON_CORE
    reason: >-
      This is a downstream morphogenesis consequence rather than the core ER
      molecular function.
    supported_by:
    - reference_id: PMID:17606994
      supporting_text: >-
        Rot1 is necessary to polarize actin cytoskeleton to the neck region at
        the end of mitosis.
- term:
    id: GO:0035269
    label: protein O-linked glycosylation via mannose
  evidence_type: IGI
  original_reference_id: PMID:22492205
  review:
    summary: >-
      Rot1 perturbation affects O-mannosylation of selected glycoproteins.
    action: KEEP_AS_NON_CORE
    reason: >-
      This is supported glycosylation biology but is best interpreted as a
      consequence of Rot1's ER chaperone/assembly role.
    supported_by:
    - reference_id: PMID:22492205
      supporting_text: >-
        Rot1 was found to be involved also in the O-mannosylation process, as
        the glycosylation of distinct glycoproteins of this type were affected.
- term:
    id: GO:0051082
    label: unfolded protein binding
  evidence_type: IDA
  original_reference_id: PMID:18508919
  review:
    summary: >-
      Direct antiaggregation data support chaperone activity, but the binding
      term is too generic.
    action: MODIFY
    reason: >-
      Replace with GO:0044183 protein folding chaperone to capture the
      functional chaperone activity.
    proposed_replacement_terms:
    - id: GO:0044183
      label: protein folding chaperone
    supported_by:
    - reference_id: PMID:18508919
      supporting_text: >-
        Recombinant Rot1 exhibited antiaggregation activity in vitro, which was
        partly impaired by a temperature-sensitive rot1-2 mutation.
- term:
    id: GO:0051082
    label: unfolded protein binding
  evidence_type: IMP
  original_reference_id: PMID:18508919
  review:
    summary: >-
      Mutant evidence supports impaired protein-folding support, not merely
      passive unfolded-protein binding.
    action: MODIFY
    reason: >-
      GO:0044183 is the better functional term for Rot1's chaperone role.
    proposed_replacement_terms:
    - id: GO:0044183
      label: protein folding chaperone
    supported_by:
    - reference_id: PMID:18508919
      supporting_text: >-
        Rot1 functions as a chaperone in vivo supporting the folding of those
        proteins.
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO terms
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000044
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt
  findings: []
- id: PMID:14562095
  title: Global analysis of protein localization in budding yeast.
  findings: []
- id: PMID:16567426
  title: Saccharomyces cerevisiae Rot1p is an ER-localized membrane protein that may function with BiP/Kar2p in protein folding.
  findings:
  - statement: Rot1 is an ER-lumen-facing membrane protein that cooperates with Kar2/BiP.
    supporting_text: >-
      Rot1p was found to be an essential, ER-localized membrane protein facing
      the lumen.
- id: PMID:16874095
  title: Causal links between protein folding in the ER and events along the secretory pathway.
  findings: []
- id: PMID:17606994
  title: Rot1 plays an antagonistic role to Clb2 in actin cytoskeleton dynamics throughout the cell cycle.
  findings: []
- id: PMID:18508919
  title: Saccharomyces cerevisiae Rot1 is an essential molecular chaperone in the endoplasmic reticulum.
  findings:
  - statement: Rot1 functions as an ER molecular chaperone.
    supporting_text: >-
      Recombinant Rot1 exhibited antiaggregation activity in vitro, and in vivo
      the rot1-2 mutation caused accelerated degradation of proteins in the
      secretory pathway.
- id: PMID:22492205
  title: The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast.
  findings:
  - statement: Rot1 is required for efficient N- and O-glycosylation.
    supporting_text: >-
      OST activity in vitro determined in membranes from rot1-1ost3Delta cells
      was found to be decreased to 45% compared with wild-type membranes.
- id: PMID:26928762
  title: 'One library to make them all: streamlining the creation of yeast libraries via a SWAp-Tag strategy.'
  findings: []
- id: PMID:9545237
  title: Cell wall integrity modulates RHO1 activity via the exchange factor ROM2.
  findings: []
- id: UniProt:Q03691
  title: UniProt entry for ROT1
  findings:
  - statement: UniProt summarizes ROT1 as an ER membrane protein involved in protein folding machinery.
    supporting_text: >-
      Involved in a protein folding machinery chaperoning proteins acting in
      various physiological processes including cell wall synthesis.
- id: file:yeast/ROT1/ROT1-deep-research-falcon.md
  title: Falcon deep research report for ROT1
  findings:
  - statement: Falcon synthesis supports Rot1 as an ER chaperone/assembly factor linked to glycoprotein maturation.
    supporting_text: >-
      Rot1's primary function is best described as an ER chaperone/assembly
      factor required for efficient glycosylation rather than a catalytic enzyme
      with a discrete small-molecule substrate.
- id: file:interpro/panther/PTHR28090/PTHR28090-metadata.yaml
  title: PANTHER family PTHR28090 ROT1 metadata
  findings:
  - statement: PTHR28090 is a ROT1-specific family supporting conserved Rot1 identity.
    supporting_text: PANTHER family PTHR28090 is named PROTEIN ROT1.
core_functions:
- molecular_function:
    id: GO:0044183
    label: protein folding chaperone
  directly_involved_in:
  - id: GO:0034975
    label: protein folding in endoplasmic reticulum
  - id: GO:0006458
    label: '''de novo'' protein folding'
  - id: GO:0006487
    label: protein N-linked glycosylation
  locations:
  - id: GO:0005789
    label: endoplasmic reticulum membrane
  description: >-
    Rot1 is an essential ER membrane chaperone or chaperone-associated assembly
    factor that supports folding and maturation of newly synthesized secretory
    and membrane proteins. It cooperates with Kar2/BiP and with the
    Ost3-containing glycosylation machinery, preventing degradation or
    underglycosylation of a subset of ER clients. N-glycosylation is therefore
    a direct core process coupled to Rot1-dependent ER proteostasis, while
    cell-wall, actin-polarity, and bud-growth phenotypes are downstream
    readouts.
  supported_by:
  - reference_id: PMID:18508919
    supporting_text: >-
      Recombinant Rot1 exhibited antiaggregation activity in vitro, which was
      partly impaired by a temperature-sensitive rot1-2 mutation.
  - reference_id: PMID:18508919
    supporting_text: >-
      Rot1 functions as a chaperone in vivo supporting the folding of those
      proteins.
  - reference_id: file:yeast/ROT1/ROT1-deep-research-falcon.md
    supporting_text: >-
      Rot1's primary function is best described as an ER chaperone/assembly
      factor required for efficient glycosylation rather than a catalytic enzyme
      with a discrete small-molecule substrate.
  - reference_id: file:interpro/panther/PTHR28090/PTHR28090-metadata.yaml
    supporting_text: PANTHER family PTHR28090 is named PROTEIN ROT1, supporting that the reviewed protein belongs to the conserved Rot1 family.
proposed_new_terms: []
suggested_questions:
- question: >-
    Is Rot1 primarily a client-binding ER chaperone, an OST/PMT assembly factor,
    or a factor that couples both activities during glycoprotein maturation?
- question: >-
    Which Rot1-dependent clients directly cause the cell wall and actin-polarity
    phenotypes when they fail to mature correctly?
suggested_experiments:
- experiment_type: client proteomics
  hypothesis: >-
    Rot1 has a restricted client set enriched for secretory-pathway and
    cell-wall glycoproteins.
  description: >-
    Compare ER-associated degradation substrates, glycosylation occupancy, and
    Rot1 proximity-labeling partners in wild-type, rot1 temperature-sensitive,
    and complemented strains under permissive and restrictive conditions.
- experiment_type: glycosylation complex reconstitution
  hypothesis: >-
    Rot1 directly improves Ost3-containing OST function through physical
    association with Ost3 rather than only indirectly through global ER stress.
  description: >-
    Reconstitute Ost3-containing and Ost6-containing OST complexes with or
    without purified Rot1 in microsomal or proteoliposome assays and measure
    substrate-specific N-glycosylation efficiency.

ROT1

Gene Description

Existing Annotations Review

Core Functions

References

Suggested Questions for Experts

Suggested Experiments

📚 Additional Documentation

Deep Research Falcon

Question

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

Target Gene/Protein Identity (from UniProt):

MANDATORY VERIFICATION STEPS:

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

Research Target:

Output

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

Target Gene/Protein Identity (from UniProt):

MANDATORY VERIFICATION STEPS:

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

Research Target:

Research Report: Saccharomyces cerevisiae ROT1 (YMR200W; UniProt Q03691)

Executive summary

Identity verification (mandatory)

1) Key concepts and definitions (current understanding)

Rot1 as an ER chaperone/assembly factor (definition)

N-glycosylation via OST and the Ost3/Ost6 isoforms (definition)

O-mannosylation (definition)

2) Functional annotation: molecular function, processes, and pathways

2.1 Subcellular localization and topology

2.2 Primary molecular function (best-supported)

2.3 Pathway context

3) Key experimental evidence (high-specificity) supporting function

3.1 Genetic interaction specificity with OST3 (not OST6)

3.2 In vivo glycosylation readouts (substrate-level)

3.3 Enzymatic activity assays: OST and PMT

3.4 Physical interaction between Rot1 and Ost3

4) Recent developments (prioritizing 2023–2024)

4.1 Rot1 as a UPR-independent ER chaperone tool (2024)

4.2 Expert framing/interpretation in 2024 work

5) Current applications and real-world implementations

5.1 Functional genomics / proteostasis engineering

5.2 Antifungal-relevant biology (contextual)

6) Relevant statistics and quantitative data (from retrieved studies)

Evidence map (quick-reference)

Conclusions for functional annotation

Key references (retrieved in this analysis)

Citations

📄 View Raw YAML