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Ref(2)P/p62 protein pleomorphism confers tissue-specific function

Yan Yan, Kai Zhang, Gabrielle Pycior, Pablo Adrian Guillen-PozaiD, Rubén HervásiD, Paulo Leal, Kexi Yi, Zulin YuiD, Ying Zhang, Laurence A. FlorensiD, Jay R. UnruhiD, Kausik SiiD

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Abstract

Protein structure dictates function and proteins often perform the same function in different cell or tissue-type. Here, we report that in Drosophila melanogaster, the broadly expressed protein Ref(2)P, an orthologue of mammalian p62/SQSTM1, performs mutually exclusive functions across tissues - autophagy in midgut and mitochondria-associated proteostasis in muscle. These functional differences arise from tissue-specific secondary and tertiary structures adopted by the same Ref(2)P polypeptide, rather than protein abundance, isoforms, post-translational modification, or binding-partner availability. The tissue-specific structures are cell autonomous and resistant to environmental perturbation. Deletion of a short intrinsically disordered region converts muscle Ref(2)P to a midgut-like structural and functional state. However, this forced switch causes muscle dysfunction, linking tissue-specific physiology to alternative structural states of the same polypeptide. We posit that Drosophila Ref(2)P exemplifies a mechanism for tissue-specificity, protein pleomorphism, in which the intrinsic structural plasticity of a protein and the physicochemical/molecular environment of a tissue enable a broadly expressed protein to adopt tissue-specific structure and function.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (5 Oct 2026) · CC BY 4.0

One Drosophila Ref(2)P polypeptide takes different structures in midgut and muscle, driving autophagy or mitochondria-associated degradation; deleting disordered region IDR2 switches muscle Ref(2)P to midgut state.

The paper notes that broadly expressed proteins are generally assumed to do the same thing in every tissue, and that whether shared proteins contribute to tissue-specific biology is unexplored. It proposes that one sequence can adopt tissue-specific structures and functions without changes in abundance, isoform or modification.

Key results

  1. ShownRef(2)P/ubiquitin puncta sit in different compartments by tissue: autophagic structures in midgut, mitochondria in flight muscle, cytosolic granules in oenocytes. Protein purified from each tissue keeps its binding preference in vitro, while E. coli protein binds both Atg8a and mitochondria.

    Localization quantified in n ≥ 9 flies per tissue. Midgut-derived protein bound Atg8a more efficiently than muscle-derived protein; muscle-derived protein bound isolated mitochondria more efficiently than midgut-derived protein. Transgene expression recapitulated localization. · Drosophila Ref(2)P protein displays distinct…; Figure legends

  2. SuggestedIn muscle, Ref(2)P loss reduces mitochondrial ubiquitin/proteasome accumulation after heat shock and heat-shock survival; genetic tests suggest mitochondria-associated degradation rather than canonical mitophagy. In midgut Ref(2)P supports autophagy.

    Ubiquitin/proteasome accumulation on muscle mitochondria after heat shock was about 42-fold in wild type vs about 1.8-fold in Ref(2)P-null. Survival after repeated heat shock was about 28% in null flies vs about 82% in controls. PINK1/Parkin loss gave no mitophagy-type phenotype; proteasome knockdown raised ubiquitin puncta. · Ref(2)P mediates autophagic degradation in…; Figure legends

  3. ShownDeleting IDR2 (amino acids 360-449) switches muscle Ref(2)P toward autophagy and damages muscle. IDR2 is not a mitochondrial targeting signal, and a stable, prevalent known post-translational modification or a difference in oligomeric state is unlikely to explain the tissue difference.

    Only ΔIDR2 raised the Ref(2)P-Atg8a interaction in muscle, and it caused loss of muscle fibers. Only about 0.1% of detected peptides in muscle Ref(2)P carried a known modification. Size-exclusion chromatography and negative-stain electron microscopy showed no oligomeric difference between tissues. · Deletion of a single IDR switches the…; IDR2-dependent functional switch is not due to…

  4. SuggestedMidgut and muscle Ref(2)P differ in secondary and tertiary structure. Crosslinking mass spectrometry plus AlphaFold-based modelling gave an 'N-C' form in midgut and an 'N-I' form in muscle. Muscle IDR2 self-assembles and reduces Atg8a binding.

    Aggregation temperature about 40 °C (midgut) vs about 55 °C (muscle); ΔIDR2 muscle protein about 54 °C vs more than 65 °C for wild-type muscle. 22 of 42 unique in-solution crosslink pairs were also found in monomers. Added muscle IDR2 significantly reduced Ref(2)P-Atg8a binding (n = 3). · Ref(2)P from muscle and midgut differs in both…; Tissue-specific three-dimensional models of…; Experimental validation of the tissue-specific…

  5. ShownThe tissue-specific route changes how aggregation-prone proteins are cleared. Htt-Q96 is handled via Ref(2)P-dependent autophagy in midgut but not muscle, and ΔIDR2 shifts muscle toward clearance.

    Ref(2)P knockdown raised Htt-Q96 about threefold in midgut with no detectable effect in muscle. Ref(2)PΔIDR2 lowered Htt-Q96 about fivefold in muscle. Aβ42 and hSOD1-A4V produced Ref(2)P aggregates in muscle (about 17-fold and 8-fold) but not midgut. · Tissue-specific function of Ref(2)P protein…

Methods, data and reproducibility

Combined Drosophila genetics (null allele, tissue-specific RNAi, domain and IDR deletions), imaging and electron microscopy, and in vitro assays on Ref(2)P purified from five sources. Structure was probed with circular dichroism, thermal and protease assays, and crosslinking mass spectrometry feeding AlphaFold2/3, AlphaLink and Rosetta models.

  • Adult tissue imaging and western blots (midgut, indirect flight muscle, oenocytes) for localization, function and genetic tests · Drosophila melanogaster · n = n ≥ 9 flies per tissue (localization); n ≥ 6 to 8 per condition (puncta)
  • Ref(2)P-sfGFP-Flag purified from E. coli, S2 cells, midgut, oenocytes and muscle; crosslinking and PTM mass spectrometry · Drosophila melanogaster (protein also expressed in E. coli) · MassIVE MSV000097980
  • Tissue RNA-seq and TMT quantitative proteomics of muscle and midgut · Drosophila melanogaster · n = three biological replicates (RNA-seq)
  • Ref(2)P-TurboID proximity labelling in muscle · Drosophila melanogaster · n = 115 enriched proteins, 31 mitochondrial
  • Heat-shock survival of control and Ref(2)P-null flies · Drosophila melanogaster · n = three independent cohorts
Methods & tools
AlphaFold2 (ColabFold), AlphaFold3, AlphaLink, Rosetta, Proteome Discoverer with XlinkX, DSSO and DMTMM crosslinkers, BeStSel, CryoSPARC, ChimeraX, STAR, RSEM, Fiji
Software & environment
Proteome Discoverer v3.0 with XlinkX v3.0; Rosetta 3.37; CryoSPARC v4.7.0; STAR 2.7.3a; RSEM v1.3.0; Huygens 14.10. No container or lockfile stated.
Assay conditions & compute
AlphaFold2 and Rosetta ran on GPU clusters of the authors' institute computation center; runtimes and memory not given.
Reproducibility gaps
Mass spectrometry raw data and search results are on MassIVE (MSV000097980). No accession is given for RNA-seq or TMT proteomics. The code covers structure modelling only.
Limitations (4)
  • Structural evidence is low resolution: no atomic-level understanding of the tissue differences, partly because small proteins with disordered regions are hard to analyze. Cryo-EM gave a high-resolution structure only for the PB1 domain, not the rest of the protein. · stated by the authors
  • How Ref(2)P engages mitochondria is unresolved. Translocation through TOM/TIM is not shown, so import with internal degradation cannot be distinguished from surface retention. The mitochondrial binding partner remains unidentified. · stated by the authors
  • Whether the pathway bias is conserved in mammals is untested, and what causes the tissue-specific structure remains unclear. · stated by the authors
  • The structural models rest on a modest set of crosslinks, AlphaFold predictions and overexpressed sfGFP-tagged protein. Only one protein in one organism, mainly two tissues, is examined. · noted in this summary

Read this if you study tissue-specific proteostasis, p62/SQSTM1 biology or fold-switching, or want a crosslink-restrained AlphaFold modelling workflow applied to a disordered-region protein.

Citations
2

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