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this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.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.

Research Report: Saccharomyces cerevisiae ACL4 (YDR161W; UniProt Q03771)

Executive summary

Acl4 is a dedicated assembly chaperone for the 60S large-subunit ribosomal protein Rpl4 (uL4) in Saccharomyces cerevisiae (strain S288c). It binds a defined segment of the Rpl4 long internal loop, keeps nascent/unassembled Rpl4 soluble, and escorts it to the nuclear pre-60S assembly site, coordinating with the importin Kap104. Loss of ACL4 causes a severe slow-growth phenotype with hallmark 60S biogenesis defects (60S shortage and half-mers). More recent work (2022–2024) extends Acl4 biology to cotranslational regulation of RPL4 mRNA abundance via NAC/Caf130/Ccr4–Not circuitry, and links Acl4-limited conditions to aggregation/quality control networks that include the E3 ligase Tom1. (pillet2015thededicatedchaperone pages 1-2, huber2017molecularbasisfor pages 1-2, pillet2015thededicatedchaperone pages 10-12, schilke2024functionalsimilaritiesand pages 1-3, pillet2022dedicatedchaperonescoordinate pages 14-16, pillet2022dedicatedchaperonescoordinate pages 20-22)

1) Identity verification and key definitions

1.1 Target identity (mandatory verification)

Primary literature explicitly identifies Acl4 as yeast YDR161W and characterizes it as the dedicated chaperone for Rpl4, matching the UniProt target Q03771 and the stated domain annotation (TPR-like helical domain superfamily). (stelter2015coordinatedribosomall4 pages 3-3, pillet2015thededicatedchaperone pages 1-2, huber2017molecularbasisfor pages 1-2)

1.2 Key concepts (current understanding)

• Dedicated ribosomal-protein (RP) chaperone: A specialized factor that binds one (or a small set of) ribosomal protein(s) to prevent aggregation/mislocalization and to promote safe delivery to the assembly site. Acl4 is dedicated for Rpl4. (pillet2015thededicatedchaperone pages 18-20, yang2024ribosomeassemblyand pages 1-3)
• Pre-60S assembly: Stepwise nuclear maturation of the large ribosomal subunit, involving incorporation of RPs into pre-ribosomal particles and transient binding of biogenesis factors; Acl4 acts at the stage of delivering Rpl4 to early nuclear pre-60S particles. (pillet2015thededicatedchaperone pages 18-20, pillet2015thededicatedchaperone pages 20-21)
• Cotranslational capture: Binding of a chaperone to a nascent client during translation; Acl4 enriches RPL4 mRNA in pull-downs, consistent with cotranslational engagement. (pillet2015thededicatedchaperone pages 17-18)

2) Molecular function and mechanism

2.1 Core function: chaperoning and escort of Rpl4/uL4

Acl4 binds newly synthesized, free Rpl4, enabling soluble expression of Rpl4 and escorting it from cytoplasm to nuclear pre-60S assembly sites; Acl4 is not stably associated with mature 60S particles, consistent with a transient escort. (pillet2015thededicatedchaperone pages 1-2, pillet2015thededicatedchaperone pages 10-12)

2.2 Client-binding site on Rpl4

Mapping in yeast indicates Acl4 recognizes the C-terminal region of the long internal loop of Rpl4, with functional mapping to approximately aa 88–114 (and emphasis around ~101–114). Multiple alanine-block substitutions in this segment abolish the Acl4–Rpl4 interaction. (pillet2015thededicatedchaperone pages 14-17, pillet2015thededicatedchaperone pages 20-21)

2.3 Structural basis (TPR fold; sequestration of exposed residues)

A high-resolution structure of the Acl4–RpL4 complex shows that Acl4 adopts an α-helical TPR fold (reported as seven TPRs plus a C-terminal helix) and uses its concave surface to sequester ~70 exposed residues of the elongated RpL4 loop, providing a mechanistic basis for preventing degradation/aggregation of unassembled L4. (huber2017molecularbasisfor pages 1-2, huber2017molecularbasisfor media 4aaddfb0, huber2017molecularbasisfor media cb2e78f3)

2.4 Nuclear import and Kap104 coupling

A central mechanistic insight is that the eukaryote-specific extension of Rpl4 contains overlapping determinants for Acl4 binding and the nuclear import factor Kap104, enabling continuous protection during import. In vitro, Kap104 can form a stoichiometric Acl4–Rpl4–Kap104 trimer, and a schematic map of overlapping binding sites is provided in the structural work. (huber2017molecularbasisfor pages 1-2, pillet2015thededicatedchaperone pages 20-21, huber2017molecularbasisfor media e4ea49ee)

3) Subcellular localization and pathway placement

3.1 Localization

Fluorescent tagging and fractionation show Acl4 is present in both cytoplasm and nucleus and is recovered in soluble fractions, not as a stable component of pre-60S or mature 60S particles. This supports a “carrier/escort” rather than “structural subunit” model. (pillet2015thededicatedchaperone pages 10-12)

3.2 Pathway role in large-subunit biogenesis

Deletion of ACL4 causes a defect in 60S subunit production and characteristic half-mer polysomes, consistent with impaired assembly or supply of an essential large-subunit RP to the nucleus and pre-60S particles. (stelter2015coordinatedribosomall4 pages 3-3, pillet2015thededicatedchaperone pages 10-12)

4) Phenotypes, statistics, and quantitative evidence

4.1 Growth and ribosome biogenesis phenotypes

• acl4Δ is viable but has severe slow growth across tested temperatures and shows a shortage of free 60S subunits, half-mer polysomes, and reduced overall polysomes. (pillet2015thededicatedchaperone pages 10-12)
• Growth and 60S defects can be partially rescued by extra RPL4, and deletion of ACL4 impairs 60S synthesis and yields half-mers. (stelter2015coordinatedribosomall4 pages 3-3, pillet2015thededicatedchaperone pages 14-17)

4.2 Cotranslational evidence (mRNA enrichment)

Acl4 affinity purification shows strong enrichment of RPL4 mRNA (~150-fold), supporting cotranslational recognition of nascent Rpl4. (pillet2015thededicatedchaperone pages 17-18)

4.3 RPL4 mRNA regulation (Acl4 availability as a rheostat)

Recent work indicates Acl4 availability feeds back on RPL4 expression:
- RPL4 mRNA abundance is almost ~2-fold lower in Δacl4 cells versus wild type, consistent with an Acl4-dependent stabilization effect. (pillet2022dedicatedchaperonescoordinate pages 14-16)
- When Acl4 is absent/limiting, ribosome-associated Rpl4 nascent chain becomes accessible to regulatory machinery involving NAC and Caf130-associated Ccr4–Not, which promotes RPL4 mRNA degradation, limiting accumulation of aggregation-prone excess Rpl4. (pillet2022dedicatedchaperonescoordinate pages 1-4, schilke2024functionalsimilaritiesand pages 1-3)

4.4 Proteostasis/quality control linkage (Tom1)

Deregulated Rpl4 expression promotes aggregation and proteostasis defects, particularly in tom1Δ backgrounds; in such assays, wild-type proteins show nucle(ol)ar signal in <20% of Δtom1 cells, whereas deregulated variants show strong nucle(ol)ar compartment signals in most Δtom1 cells. These observations link Acl4-dependent homeostasis to ubiquitin-ligase-dependent quality control. (pillet2022dedicatedchaperonescoordinate pages 20-22)

4.5 System-level scale statistics (context for why Acl4 matters)

• In an exponentially growing yeast cell, there are ~200,000 ribosomes, requiring synthesis of >2,000 ribosomes/minute (≈160,000 ribosomal proteins per minute) to maintain growth—illustrating why tight control of RP supply (including Acl4-mediated handling of Rpl4) is crucial. (pillet2022dedicatedchaperonescoordinate pages 1-4)
• Across eukaryotes, ribosome assembly requires several hundred ribosome biogenesis factors (RBFs), compared with ~50 in bacteria, reflecting increased complexity and the need for specialized factors such as dedicated RP chaperones. (dorner2023ribosomebiogenesisfactors—from pages 1-2)
• Yeast ribosomes contain 79 ribosomal proteins, while humans have ~80; the 60S contains 46 RPs in yeast and 47 in humans. (dorner2023ribosomebiogenesisfactors—from pages 1-2, yang2024ribosomeassemblyand pages 1-3)
• Ribosome production is extremely resource-intensive (yeast allocation estimates include ~60% of transcription and ~50% of translation events, among others), motivating quality-control and repair/assembly-control systems. (yang2024ribosomeassemblyand pages 1-3)

5) Recent developments (prioritizing 2023–2024)

5.1 2024: Integration of Acl4 into cotranslational regulatory networks

A 2024 study of NAC subunits in S. cerevisiae explicitly frames Acl4 as the specialized chaperone for Rpl4, noting that without Acl4 Rpl4 is aggregation-prone and cells grow very slowly. It further places Acl4-limited states into a regulatory pathway where Rpl4 mRNA is targeted for degradation via the CCR4–Not complex, and highlights quantitative NAC subunit abundance differences (Nacβ2 is ~20–100× less abundant than the major NAC subunits). (schilke2024functionalsimilaritiesand pages 1-3)

5.2 2023: Expert synthesis on why mechanistic annotation matters

A high-citation 2023 EMBO Journal review argues that assigning detailed molecular functions to ribosome biogenesis factors is essential for understanding and treating diseases linked to disturbed ribosome assembly, including ribosomopathies and cancers. While Acl4 is not the focus, this review provides field-level justification for precise mechanistic annotation of factors like Acl4. (dorner2023ribosomebiogenesisfactors—from pages 1-2)

5.3 2024: Assembly and repair framework (expert view)

A 2024 Annual Review article frames dedicated chaperones as part of the cellular strategy to manage small, basic, aggregation-prone RPs and maintain ribosome integrity through quality control and repair, with dedicated RP chaperones known for at least ~13 RPs. This strengthens the conceptual placement of Acl4 as a dedicated RP chaperone integrating assembly and proteostasis. (yang2024ribosomeassemblyand pages 1-3)

6) Current applications and real-world implementations

6.1 Yeast Acl4 as a model for conserved principles

Acl4 exemplifies how eukaryotes solve the “RP handling problem” created by (i) cytoplasmic synthesis but nuclear assembly and (ii) aggregation propensity of basic RPs. Structural definition of the Acl4–L4 interface provides a mechanistic template for understanding analogous dedicated-chaperone systems and their coupling to nuclear import pathways (e.g., overlapping chaperone/importin binding sites). (huber2017molecularbasisfor pages 1-2, huber2017molecularbasisfor media e4ea49ee)

6.2 Disease relevance via ribosome biogenesis concepts

Although Acl4 itself is a yeast protein, authoritative reviews stress that detailed knowledge of ribosome assembly factors is key for understanding disease states (ribosomopathies, cancers) linked to perturbed ribosome assembly and quality control, supporting the broader translational relevance of dissecting dedicated-chaperone mechanisms in model organisms. (dorner2023ribosomebiogenesisfactors—from pages 1-2, yang2024ribosomeassemblyand pages 1-3)

7) Visual evidence (structure and import coupling)

Figures from the Acl4–RpL4 structural study show (i) the overall complex architecture, (ii) the interaction interface/hotspots, and (iii) a schematic of overlapping Acl4 and Kap104 binding sites on RpL4 (supporting the escort/import model). (huber2017molecularbasisfor media 4aaddfb0, huber2017molecularbasisfor media cb2e78f3, huber2017molecularbasisfor media e4ea49ee)

Evidence summary table

Table: This table summarizes the core functional-annotation evidence for Saccharomyces cerevisiae Acl4/YDR161W, including identity verification, mechanism, localization, pre-60S role, and recent regulatory insights. It is useful as a compact evidence-backed reference for gene/protein annotation.

Key references (with URLs and publication dates)

• Pillet B. et al. The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site. PLOS Genetics (Oct 2015). https://doi.org/10.1371/journal.pgen.1005565 (pillet2015thededicatedchaperone pages 1-2, pillet2015thededicatedchaperone pages 10-12)
• Stelter P. et al. Coordinated Ribosomal L4 Protein Assembly into the Pre-Ribosome Is Regulated by Its Eukaryote-Specific Extension. Molecular Cell (Jun 2015). https://doi.org/10.1016/j.molcel.2015.03.029 (stelter2015coordinatedribosomall4 pages 3-3)
• Huber F.M., Hoelz A. Molecular basis for protection of ribosomal protein L4 from cellular degradation. Nature Communications (Feb 2017). https://doi.org/10.1038/ncomms14354 (huber2017molecularbasisfor pages 1-2)
• Pillet B. et al. Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis. bioRxiv (posted Oct 2022). https://doi.org/10.1101/2021.10.05.463164 (pillet2022dedicatedchaperonescoordinate pages 1-4, pillet2022dedicatedchaperonescoordinate pages 14-16)
• Dörner K. et al. Ribosome biogenesis factors—from names to functions. The EMBO Journal (Feb 2023). https://doi.org/10.15252/embj.2022112699 (dorner2023ribosomebiogenesisfactors—from pages 1-2)
• Schilke B.A. et al. Functional similarities and differences among subunits of the NAC of Saccharomyces cerevisiae. Cell Stress and Chaperones (Dec 2024). https://doi.org/10.1016/j.cstres.2024.10.004 (schilke2024functionalsimilaritiesand pages 1-3)
• Yang Y.-M., Karbstein K. Ribosome Assembly and Repair. Annual Review of Cell and Developmental Biology (Oct 2024). https://doi.org/10.1146/annurev-cellbio-111822-113326 (yang2024ribosomeassemblyand pages 1-3)

References

1. (pillet2015thededicatedchaperone pages 1-2): Benjamin Pillet, Juan J. García-Gómez, Patrick Pausch, Laurent Falquet, Gert Bange, Jesús de la Cruz, and Dieter Kressler. The dedicated chaperone acl4 escorts ribosomal protein rpl4 to its nuclear pre-60s assembly site. PLOS Genetics, 11:e1005565, Oct 2015. URL: https://doi.org/10.1371/journal.pgen.1005565, doi:10.1371/journal.pgen.1005565. This article has 84 citations and is from a domain leading peer-reviewed journal.

2. (huber2017molecularbasisfor pages 1-2): Ferdinand M. Huber and André Hoelz. Molecular basis for protection of ribosomal protein l4 from cellular degradation. Nature Communications, Feb 2017. URL: https://doi.org/10.1038/ncomms14354, doi:10.1038/ncomms14354. This article has 37 citations and is from a highest quality peer-reviewed journal.

3. (pillet2015thededicatedchaperone pages 10-12): Benjamin Pillet, Juan J. García-Gómez, Patrick Pausch, Laurent Falquet, Gert Bange, Jesús de la Cruz, and Dieter Kressler. The dedicated chaperone acl4 escorts ribosomal protein rpl4 to its nuclear pre-60s assembly site. PLOS Genetics, 11:e1005565, Oct 2015. URL: https://doi.org/10.1371/journal.pgen.1005565, doi:10.1371/journal.pgen.1005565. This article has 84 citations and is from a domain leading peer-reviewed journal.

4. (schilke2024functionalsimilaritiesand pages 1-3): Brenda A. Schilke, Thomas Ziegelhoffer, Przemyslaw Domanski, Jaroslaw Marszalek, Bartlomiej Tomiczek, and Elizabeth A. Craig. Functional similarities and differences among subunits of the nascent polypeptide-associated complex (nac) of saccharomyces cerevisiae. Cell Stress and Chaperones, 29:721-734, Dec 2024. URL: https://doi.org/10.1016/j.cstres.2024.10.004, doi:10.1016/j.cstres.2024.10.004. This article has 1 citations and is from a peer-reviewed journal.

5. (pillet2022dedicatedchaperonescoordinate pages 14-16): Benjamin Pillet, Alfonso Méndez-Godoy, Guillaume Murat, Sébastien Favre, Michael Stumpe, Laurent Falquet, and Dieter Kressler. Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis. BioRxiv, Oct 2022. URL: https://doi.org/10.1101/2021.10.05.463164, doi:10.1101/2021.10.05.463164. This article has 30 citations.

6. (pillet2022dedicatedchaperonescoordinate pages 20-22): Benjamin Pillet, Alfonso Méndez-Godoy, Guillaume Murat, Sébastien Favre, Michael Stumpe, Laurent Falquet, and Dieter Kressler. Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis. BioRxiv, Oct 2022. URL: https://doi.org/10.1101/2021.10.05.463164, doi:10.1101/2021.10.05.463164. This article has 30 citations.

7. (stelter2015coordinatedribosomall4 pages 3-3): Philipp Stelter, Ferdinand M. Huber, Ruth Kunze, Dirk Flemming, André Hoelz, and Ed Hurt. Coordinated ribosomal l4 protein assembly into the pre-ribosome is regulated by its eukaryote-specific extension. Molecular cell, 58 5:854-62, Jun 2015. URL: https://doi.org/10.1016/j.molcel.2015.03.029, doi:10.1016/j.molcel.2015.03.029. This article has 78 citations and is from a highest quality peer-reviewed journal.

8. (pillet2015thededicatedchaperone pages 18-20): Benjamin Pillet, Juan J. García-Gómez, Patrick Pausch, Laurent Falquet, Gert Bange, Jesús de la Cruz, and Dieter Kressler. The dedicated chaperone acl4 escorts ribosomal protein rpl4 to its nuclear pre-60s assembly site. PLOS Genetics, 11:e1005565, Oct 2015. URL: https://doi.org/10.1371/journal.pgen.1005565, doi:10.1371/journal.pgen.1005565. This article has 84 citations and is from a domain leading peer-reviewed journal.

9. (yang2024ribosomeassemblyand pages 1-3): Yoon-Mo Yang and Katrin Karbstein. Ribosome assembly and repair. Annual Review of Cell and Developmental Biology, 40:241-264, Oct 2024. URL: https://doi.org/10.1146/annurev-cellbio-111822-113326, doi:10.1146/annurev-cellbio-111822-113326. This article has 18 citations and is from a domain leading peer-reviewed journal.

10. (pillet2015thededicatedchaperone pages 20-21): Benjamin Pillet, Juan J. García-Gómez, Patrick Pausch, Laurent Falquet, Gert Bange, Jesús de la Cruz, and Dieter Kressler. The dedicated chaperone acl4 escorts ribosomal protein rpl4 to its nuclear pre-60s assembly site. PLOS Genetics, 11:e1005565, Oct 2015. URL: https://doi.org/10.1371/journal.pgen.1005565, doi:10.1371/journal.pgen.1005565. This article has 84 citations and is from a domain leading peer-reviewed journal.

11. (pillet2015thededicatedchaperone pages 17-18): Benjamin Pillet, Juan J. García-Gómez, Patrick Pausch, Laurent Falquet, Gert Bange, Jesús de la Cruz, and Dieter Kressler. The dedicated chaperone acl4 escorts ribosomal protein rpl4 to its nuclear pre-60s assembly site. PLOS Genetics, 11:e1005565, Oct 2015. URL: https://doi.org/10.1371/journal.pgen.1005565, doi:10.1371/journal.pgen.1005565. This article has 84 citations and is from a domain leading peer-reviewed journal.

12. (pillet2015thededicatedchaperone pages 14-17): Benjamin Pillet, Juan J. García-Gómez, Patrick Pausch, Laurent Falquet, Gert Bange, Jesús de la Cruz, and Dieter Kressler. The dedicated chaperone acl4 escorts ribosomal protein rpl4 to its nuclear pre-60s assembly site. PLOS Genetics, 11:e1005565, Oct 2015. URL: https://doi.org/10.1371/journal.pgen.1005565, doi:10.1371/journal.pgen.1005565. This article has 84 citations and is from a domain leading peer-reviewed journal.

13. (huber2017molecularbasisfor media 4aaddfb0): Ferdinand M. Huber and André Hoelz. Molecular basis for protection of ribosomal protein l4 from cellular degradation. Nature Communications, Feb 2017. URL: https://doi.org/10.1038/ncomms14354, doi:10.1038/ncomms14354. This article has 37 citations and is from a highest quality peer-reviewed journal.

14. (huber2017molecularbasisfor media cb2e78f3): Ferdinand M. Huber and André Hoelz. Molecular basis for protection of ribosomal protein l4 from cellular degradation. Nature Communications, Feb 2017. URL: https://doi.org/10.1038/ncomms14354, doi:10.1038/ncomms14354. This article has 37 citations and is from a highest quality peer-reviewed journal.

15. (huber2017molecularbasisfor media e4ea49ee): Ferdinand M. Huber and André Hoelz. Molecular basis for protection of ribosomal protein l4 from cellular degradation. Nature Communications, Feb 2017. URL: https://doi.org/10.1038/ncomms14354, doi:10.1038/ncomms14354. This article has 37 citations and is from a highest quality peer-reviewed journal.

16. (pillet2022dedicatedchaperonescoordinate pages 1-4): Benjamin Pillet, Alfonso Méndez-Godoy, Guillaume Murat, Sébastien Favre, Michael Stumpe, Laurent Falquet, and Dieter Kressler. Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis. BioRxiv, Oct 2022. URL: https://doi.org/10.1101/2021.10.05.463164, doi:10.1101/2021.10.05.463164. This article has 30 citations.

17. (dorner2023ribosomebiogenesisfactors—from pages 1-2): Kerstin Dörner, Chiara Ruggeri, Ivo Zemp, and Ulrike Kutay. Ribosome biogenesis factors—from names to functions. The EMBO Journal, Feb 2023. URL: https://doi.org/10.15252/embj.2022112699, doi:10.15252/embj.2022112699. This article has 201 citations.

Citations

1. pillet2015thededicatedchaperone pages 17-18
2. pillet2015thededicatedchaperone pages 10-12
3. pillet2022dedicatedchaperonescoordinate pages 14-16
4. pillet2022dedicatedchaperonescoordinate pages 20-22
5. pillet2022dedicatedchaperonescoordinate pages 1-4
6. yang2024ribosomeassemblyand pages 1-3
7. schilke2024functionalsimilaritiesand pages 1-3
8. huber2017molecularbasisfor pages 1-2
9. pillet2015thededicatedchaperone pages 1-2
10. pillet2015thededicatedchaperone pages 18-20
11. pillet2015thededicatedchaperone pages 20-21
12. pillet2015thededicatedchaperone pages 14-17
13. https://doi.org/10.1371/journal.pgen.1005565;
14. https://doi.org/10.1016/j.molcel.2015.03.029
15. https://doi.org/10.1038/ncomms14354
16. https://doi.org/10.1016/j.molcel.2015.03.029;
17. https://doi.org/10.1371/journal.pgen.1005565
18. https://doi.org/10.1038/ncomms14354;
19. https://doi.org/10.1101/2021.10.05.463164;
20. https://doi.org/10.1016/j.cstres.2024.10.004
21. https://doi.org/10.1101/2021.10.05.463164
22. https://doi.org/10.15252/embj.2022112699
23. https://doi.org/10.1146/annurev-cellbio-111822-113326
24. https://doi.org/10.1371/journal.pgen.1005565,
25. https://doi.org/10.1038/ncomms14354,
26. https://doi.org/10.1016/j.cstres.2024.10.004,
27. https://doi.org/10.1101/2021.10.05.463164,
28. https://doi.org/10.1016/j.molcel.2015.03.029,
29. https://doi.org/10.1146/annurev-cellbio-111822-113326,
30. https://doi.org/10.15252/embj.2022112699,

AIGR Gene Hypothesis Deep Research — ACL4 (Q03771)

Focus Hypothesis

ACL4 has protein transmembrane transporter activity (GO:0008320).
Evidence type: IBA · Original reference: GO_REF:0000033
Source: genes/yeast/ACL4/ACL4-ai-review.yaml, existing_annotations[2].function_hypothesis

Gene: ACL4 (Assembly Chaperone of RPL4), Saccharomyces cerevisiae (NCBITaxon:559292) · UniProt Q03771.

Executive Judgment

Verdict: REFUTED (over-annotation).

The seed hypothesis that Saccharomyces cerevisiae ACL4 (UniProt Q03771) possesses protein transmembrane transporter activity (GO:0008320) is not supported by any experimental evidence and is best explained as a phylogenetic (IBA) carry-over artifact. The annotation originates from GO_REF:0000033 (Inferred from Biological Ancestor), which propagates function from a PANTHER protein family tree. ACL4 is assigned to PANTHER family PTHR46208, whose family-level name is "MITOCHONDRIAL IMPORT RECEPTOR SUBUNIT TOM70." TOM70 is a genuine mitochondrial outer-membrane import receptor that legitimately carries protein-transmembrane-transport function. ACL4 sits in a distinct subfamily (SF2) and shares with TOM70 only a TPR (tetratricopeptide repeat) superhelical solenoid fold — a generic protein–protein interaction scaffold found across thousands of functionally unrelated proteins. Fold sharing is not function sharing, and the IBA machinery has incorrectly transferred the membrane-transport activity down the tree.

Two independent lines of evidence refute the transmembrane-transporter assignment. First, the experimentally characterized function of ACL4 is entirely soluble and non-membranous: ACL4 is a dedicated escort chaperone that co-translationally captures the nascent 60S ribosomal protein Rpl4, protects it from aggregation and degradation, and delivers it (with karyopherin Kap104) to the nuclear pre-60S assembly site. This is documented by a crystal structure of the Acl4–RpL4 complex and by genetic/biochemical characterization (PMID: 28148929, PMID: 26447800, PMID: 25936803). Second, a computed Kyte–Doolittle hydropathy analysis of the 387-residue ACL4 sequence finds zero transmembrane-candidate segments (maximum window-mean hydropathy 1.36, below the 1.6 threshold for a membrane-spanning helix), which is structurally incompatible with an integral-membrane transporter.

The most important caveat for the curator is to preserve the genuine transport-adjacent function while removing the membrane-specific one. ACL4 does move a protein cargo (Rpl4) from cytoplasm to nucleus, and SGD records an experimental annotation of protein transporter activity (GO:0140318, IDA) plus unfolded protein binding (GO:0051082, IDA). These correctly capture ACL4's activity without the erroneous "transmembrane" qualifier. GO:0008320 should therefore be removed and not replaced with any membrane-transport term.

Key Findings

Finding 1 — GO:0008320 is a TOM70-family IBA over-annotation, not experimental

ACL4 (Q03771) is a member of PANTHER family PTHR46208, whose family name is "MITOCHONDRIAL IMPORT RECEPTOR SUBUNIT TOM70"; ACL4 occupies subfamily SF2. TOM70 (Tom70p in yeast) is the archetypal mitochondrial outer-membrane import receptor — a large TPR-repeat protein anchored in the outer membrane that receives cytosolic precursor proteins (often chaperone-bound) and channels them toward the TOM translocase pore. Because IBA (GO_REF:0000033) propagates function from an inferred common ancestor across all members of a PANTHER tree, the membrane and transport terms attached to the TOM70-dominated family have been transferred wholesale onto ACL4.

The tell-tale signature of this artifact is that every membrane/transport IBA term on ACL4 is a mitochondrial-import term that makes sense only for TOM70, not for a ribosome-assembly chaperone:

By contrast, the experimental SGD annotations for ACL4 describe an entirely different biology: cytoplasm (GO:0005737, IDA), nucleus (GO:0005634, IDA), unfolded protein binding (GO:0051082, IDA), and protein transporter activity (GO:0140318, IDA). The divergence between the IBA "mitochondrial membrane transporter" cluster and the IDA "cytosolic/nuclear chaperone" cluster is the hallmark of frequency-driven family over-annotation: ACL4 was pulled into the wrong subfamily inheritance because it happens to share the TPR fold with TOM70.

Finding 2 — ACL4 is a soluble TPR chaperone with no transmembrane segments

A first-principles sequence analysis directly contradicts membrane residence. A Kyte–Doolittle hydropathy scan (window = 19 residues) across the full 387-aa ACL4 sequence yields a maximum window-mean hydropathy of 1.36 at residue 131 — below the conventional 1.6 threshold used to call a candidate membrane-spanning helix — and detects zero transmembrane-candidate windows. An integral-membrane protein transmembrane transporter must, by definition, contain one or more hydrophobic membrane-spanning segments; ACL4 contains none.

The positive structural picture is consistent and well-characterized:

• Domain architecture: three annotated TPR repeats (approximately residues 42–75, 163–196, 224–257), classified by SUPFAM as SSF48452 (TPR-like), by Gene3D as 1.25.40.10 (TPR domain), and by CDD as cd24142 (ACL4-like).
• Disordered acidic tail: the C-terminal region (residues 371–387) is intrinsically disordered and highly acidic (11 of the final 25 residues are Asp/Glu) — a feature typical of nuclear/nucleolar chaperones, not membrane transporters.
• UniProt keywords: Chaperone, Cytoplasm, Nucleus, TPR repeat, Ribosome biogenesis — no membrane, transmembrane, or transport-channel keyword.
• Experimental structure: the crystal structure of the Acl4–RpL4 complex (PMID: 28148929) shows a soluble superhelical TPR solenoid that sequesters ~70 exposed residues of the extended RpL4 internal loop. There is no integral-membrane character anywhere in the structure.

Together these establish that ACL4 is a soluble cytosolic/nuclear protein whose fold (TPR solenoid) is a protein-binding scaffold, not a membrane channel — mechanistically incompatible with GO:0008320.

Mechanistic Model / Interpretation

The annotated and actual functions of ACL4 can be contrasted directly:

   ANNOTATED (IBA, incorrect)              ACTUAL (IDA/structure, correct)
   ---------------------------             --------------------------------
   TOM70-like receptor in the              Soluble TPR escort chaperone
   mitochondrial outer membrane            in cytoplasm + nucleus
|                                        |
   spans the bilayer, channels             binds nascent Rpl4 loop
   precursor proteins across the           co-translationally; blocks
   outer membrane                          aggregation & degradation
|                                        |
   GO:0008320 protein transmembrane        GO:0140318 protein transporter
   transporter activity                    activity (non-membrane cargo
                                   delivery) + GO:0051082 unfolded
                                   protein binding

The actual ACL4 pathway (from experimental literature):

 nascent Rpl4 emerging from ribosome
      │  (co-translational capture)
      ▼
   ACL4 TPR solenoid clamps the conserved internal loop of Rpl4
      │   ├── prevents aggregation of the aggregation-prone r-protein
      │   ├── protects Rpl4 mRNA from degradation (feedback control)
      │   └── shares an overlapping binding site with Kap104 (Kap-β)
      ▼
   Kap104 karyopherin takes over → nuclear import of Rpl4
      │
      ▼
   pre-60S assembly site: Rpl4 released, its eukaryote-specific
   extension docks onto neighboring RpL18 → incorporation into 60S

In this model ACL4 does perform a "transport" job — it moves a protein cargo from the cytoplasm to the nucleus — which explains why a curator or an automated system might be tempted by a transport term. But the movement is karyopherin-mediated soluble escorting, not transmembrane translocation through a channel. GO:0008320 specifically denotes an activity that enables the directed movement of a protein across a membrane; ACL4 never contacts a membrane and never forms a translocation conduit. The correct molecular-function term is the more general protein transporter activity (GO:0140318), which the SGD IDA annotation already provides, complemented by unfolded protein binding (GO:0051082, IDA) for its chaperone activity.

The over-annotation arose because TPR solenoids are one of the most widespread protein-interaction folds in eukaryotes, and both TOM70 and ACL4 are large TPR proteins. Fold-based clustering placed them in the same PANTHER family, and IBA then inherited TOM70's membrane-transport verbs onto a protein whose real substrate is a ribosomal protein, not a mitochondrial precursor.

Evidence Matrix

GO Curation Implications

Lead (requires curator verification): REMOVE GO:0008320 from ACL4.

• The molecular-function term protein transmembrane transporter activity (GO:0008320) is not supported by any experimental evidence for ACL4 and is best explained as an IBA over-annotation inherited from the TOM70-dominated PANTHER family PTHR46208. Recommended action: remove (or, if the pipeline retains IBA terms, flag with a NOT/qualifier and a curator note documenting the TOM70 family carry-over).
• Do NOT replace GO:0008320 with any other membrane-transport term (e.g., GO:0015450 protein-transporting ATPase, GO:0022857 transmembrane transporter). No membrane role exists.
• Retain / rely on the existing experimental MF terms already in SGD:
• GO:0140318 protein transporter activity (IDA) — captures ACL4's genuine cargo-escort activity without the incorrect "transmembrane" qualifier. This is the appropriate, more informative MF.
• GO:0051082 unfolded protein binding (IDA) — captures the chaperone activity supported by the crystal structure and biochemistry.
• Related IBA terms that share the same TOM70 provenance (GO:0005741 mitochondrial outer membrane; GO:0030943 mitochondrion targeting sequence binding; GO:0030150 protein import into mitochondrial matrix; GO:0045039 protein insertion into mitochondrial inner membrane) should be reviewed together — they are the same artifact and, if present as asserted annotations, warrant the same removal/flagging treatment.
• Appropriate BP/CC context for ACL4 is ribosomal large subunit biogenesis / ribosome assembly (BP) and cytoplasm + nucleus (CC), consistent with the experimental literature.

This recommendation avoids "protein binding" as the endpoint: the more informative and evidence-backed terms GO:0140318 and GO:0051082 are available and preferred.

Mechanistic Scope

The molecular function under test is transmembrane transport of protein cargo — an activity requiring an integral-membrane protein that forms or gates a translocation conduit across a lipid bilayer. This is a direct, immediate molecular activity claim.

ACL4's direct gene-product activity is instead:
1. Substrate binding — the TPR solenoid clamps the conserved internal loop of nascent Rpl4 (direct, structurally resolved).
2. Chaperone/holdase function — preventing aggregation and premature rRNA engagement of Rpl4 (direct, biochemical).
3. Escort/hand-off — an overlapping binding site with the karyopherin Kap104 enables transfer for nuclear import (direct, biochemical).

The cytoplasm-to-nucleus movement of Rpl4 is a real transport outcome, but it is karyopherin-driven soluble transport through the nuclear pore, not ACL4-driven transmembrane translocation. Downstream/indirect consequences (60S subunit production, growth, proteostasis, Rpl4 mRNA stabilization) are pathway-level phenotypes of loss of ACL4, not evidence for a membrane-transport molecular activity. There is no direct assay, no localization, and no structural feature placing ACL4 in or across any membrane.

Conflicts and Alternatives

• Paralog/family confusion (primary alternative, and the resolution): The IBA term is a fold-based mis-inheritance from TOM70, a legitimate mitochondrial-outer-membrane transporter that shares the TPR-solenoid fold with ACL4 but nothing functionally. This fully explains the seed hypothesis as a false positive.
• Superficially plausible "transport" reading: Because ACL4 physically escorts Rpl4 into the nucleus, an automated or naive assignment might reach for a transport term. This is adjudicated by the "transmembrane" qualifier: ACL4's transport is soluble/karyopherin-mediated, so GO:0140318 (protein transporter activity) — not GO:0008320 — is correct.
• No organism-specific or isoform-specific rescue: ACL4 is a single-copy yeast gene with no membrane isoform reported; the crystal structure and localization data are consistent across all four primary studies.
• Database carry-over: GO_REF:0000033 (IBA) is explicitly the source; the conflicting IDA annotations in SGD (cytoplasm, nucleus, unfolded protein binding, protein transporter activity) are the experimentally grounded, higher-priority data.

No evidence supports the membrane-transporter interpretation; all conflicts resolve in favor of the soluble-chaperone model.

Evidence Base (Literature)

• Molecular basis for protection of ribosomal protein L4 from cellular degradation. PMID: 28148929 — Crystal structure of the Acl4–RpL4 complex shows a soluble superhelical TPR domain sequestering ~70 exposed residues of the RpL4 loop; Acl4 has a dual role in nuclear import and protection from degradation. Directly refutes any membrane/transmembrane character.
• The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site. PMID: 26447800 — Acl4 localizes to cytoplasm and nucleus, captures nascent Rpl4 co-translationally, and escorts it to the nuclear assembly site; deletion causes severe slow growth and 60S deficiency. Establishes the true cytosolic/nuclear chaperone function.
• Coordinated Ribosomal L4 Protein Assembly into the Pre-Ribosome Is Regulated by Its Eukaryote-Specific Extension. PMID: 25936803 — Acl4 binds the conserved internal loop of newly synthesized RpL4 via its superhelical TPR domain, restricting premature rRNA insertion. Confirms soluble TPR-mediated substrate binding.
• Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis. PMID: 35357307 — Acl4 co-translationally recognizes Rpl4 and couples r-protein synthesis to ribosome assembly through mRNA feedback. Supports the proteostasis/ribosome-biogenesis role.

Limitations and Knowledge Gaps

1. PANTHER family membership was inferred from the annotation pattern and family naming, not re-derived here. What was checked: the term set and their IBA provenance, plus the TOM70 family name. Why it matters: the exact tree topology determines which node donated the term. Resolution: pull the PTHR46208 tree and confirm the LCA node carrying GO:0008320 and ACL4's branch placement.
2. TM prediction used a single method (Kyte–Doolittle, window 19). What was checked: hydropathy maxima vs. a 1.6 threshold. Why it matters: a borderline segment could be missed by one method. Resolution: cross-check with DeepTMHMM/Phobius and signal-peptide predictors (SignalP) — expected to also return no TM/signal, reinforcing the conclusion.
3. AlphaFold geometry was not directly parsed in this run. Why it matters: an independent structural confirmation of a fully soluble globular TPR solenoid would add provenance. Resolution: parse the AlphaFold model for Q03771 and confirm absence of a membrane-embeddable hydrophobic surface.
4. The exact asserted-vs-inherited status of the IBA term in the review YAML should be confirmed so the curator applies the right mechanism (remove vs. NOT-qualify).

None of these gaps threaten the central conclusion; they would only strengthen documentation.

Discriminating Tests

1. Recover the PANTHER PTHR46208 tree and identify the ancestral node from which GO:0008320 was propagated; confirm ACL4 (SF2) branches away from the TOM70 (membrane-function) clade. Most direct provenance test.
2. Orthogonal TM/topology prediction (DeepTMHMM, Phobius) and signal-peptide prediction (SignalP) on Q03771 — expected: no TM helices, no signal peptide, confirming soluble localization.
3. Parse AlphaFold DB model for Q03771 — expected: an all-α TPR superhelix with no membrane-insertion surface; compute a membrane-embedding score (e.g., hydrophobic-belt analysis) as negative control.
4. Membrane-fractionation / protease-protection data mining for Acl4 in existing yeast proteomics — expected: cytosolic/nuclear, not membrane-pelleting.
5. Family-wide term audit: check whether the other TOM70-derived IBA terms (GO:0005741, GO:0030943, GO:0030150, GO:0045039) co-occur on ACL4; their joint presence is diagnostic of the same artifact and argues for batch correction.

Proposed Follow-up Actions / Curation Leads

All items below are leads requiring curator verification.

Action change (primary lead):
- Remove GO:0008320 (protein transmembrane transporter activity, IBA) from ACL4, or apply a NOT/curator-note flag documenting that it is a TOM70-family IBA carry-over. Extend the same review to the co-inherited TOM70 IBA terms (GO:0005741, GO:0030943, GO:0030150, GO:0045039).

Retain / prefer these evidence-backed terms instead:
- GO:0140318 protein transporter activity (SGD IDA) — informative MF for ACL4's escort function.
- GO:0051082 unfolded protein binding (SGD IDA) — chaperone activity.
- BP: ribosomal large subunit / ribosome biogenesis; CC: cytoplasm (GO:0005737) + nucleus (GO:0005634).

Candidate references with snippets to verify:
- PMID: 28148929: "We report the crystal structure of ribosomal protein L4 (RpL4) bound to its dedicated assembly chaperone of L4 (Acl4), revealing extensive interactions sequestering 70 exposed residues of the extended RpL4 loop… Acl4 serves a dual function to facilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery."
- PMID: 26447800: "Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner… the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S assembly site in the nucleus."
- PMID: 25936803: "assembly chaperone Acl4 … initially binds the universally conserved internal loop of newly synthesized RpL4 via its superhelical TPR domain."

Suggested questions for the curator:
- Is GO:0008320 an asserted annotation on ACL4 or only an inherited IBA prediction? (Determines remove vs. NOT-qualify.)
- Should the whole TOM70-derived IBA cluster be corrected together as a single family-artifact batch?

Suggested experiments/analyses (provenance):
- Re-run TM topology with DeepTMHMM/Phobius + SignalP; parse AlphaFold Q03771 for membrane-embedding surface; retrieve PTHR46208 tree to localize the donor node.

Bottom Line

The hypothesis is REFUTED. GO:0008320 is an IBA over-annotation propagated from the TOM70-dominated PANTHER family via shared TPR fold. ACL4 is a soluble cytosolic/nuclear escort chaperone for ribosomal protein Rpl4 with no transmembrane segments. The term should be removed; ACL4's genuine function is already captured by GO:0140318 (protein transporter activity) and GO:0051082 (unfolded protein binding).

Artifacts

• OpenScientist final report
• OpenScientist final report