PEX16

PEX16 is an integral peroxisomal membrane protein that serves as a central organizer of peroxisome biogenesis and maintenance by recruiting other peroxisomal membrane proteins (PEX3, PMP34) to the endoplasmic reticulum and mediating their Sec16B-dependent export to peroxisomes (PMID:16717127, PMID:21768384, PMID:25002403). PEX16 is cotranslationally inserted into the ER, where distinct functional domains govern both its own ER-to-peroxisome trafficking and its PMP recruitment activity, a function conserved in plants (PMID:25903784). PEX16 accelerates de novo peroxisomal membrane formation from the ER independently of PEX3 targeting, though it is not absolutely required, as PEX16-knockout cells retain reduced numbers of enlarged peroxisomes; loss of PEX16 also triggers compensatory pexophagy (PMID:35437598, PMID:34360754). Loss-of-function mutations in PEX16 cause Zellweger syndrome (complementation group D) by abolishing peroxisomal membrane assembly (PMID:9837814).

1. 1997 High

   Identification of Pex16p as a peroxisome-associated protein in yeast established the gene locus and showed that its overexpression enlarges peroxisomes while maintaining their protein complement, defining Pex16p as a factor controlling peroxisome size rather than import.

   Evidence Subcellular fractionation, mutagenesis, and electron microscopy in Yarrowia lipolytica

   PMID:9182661

   Open questions at the time

   • Function of the mammalian ortholog was unknown
   • Mechanism by which Pex16p controls peroxisome size was undefined
   • Topology and membrane insertion pathway were unresolved
2. 1998 High

   Cloning of human PEX16 and demonstration that its loss-of-function mutations cause Zellweger syndrome (CG-D) established the gene as essential for peroxisome biogenesis in humans and linked it to a defined Mendelian disease.

   Evidence cDNA cloning, complementation rescue in patient fibroblasts, mutation analysis

   PMID:9837814

   Open questions at the time

   • How PEX16 mechanistically contributes to peroxisome membrane assembly was unknown
   • Whether PEX16 acts at the ER or directly at peroxisomes was unresolved
   • Functional domain architecture was not mapped
3. 2006 High

   Demonstrating that PEX16 is cotranslationally inserted into the ER and recruits other PMPs there for de novo peroxisome formation resolved the long-standing question of whether new peroxisomes arise from the ER or solely by division, establishing the ER-origin pathway.

   Evidence Live-cell pulse-chase imaging with photoactivatable GFP in peroxisome-less and wild-type human cells

   PMID:16717127

   Open questions at the time

   • The ER export machinery mediating PEX16 transport to peroxisomes was unidentified
   • Whether PEX16 also functions in constitutive peroxisome maintenance was untested
   • Which PMPs are directly recruited by PEX16 at the ER was not systematically defined
4. 2011 High

   Identification of Sec16B as required for ER-to-peroxisome export of PEX16 and PEX3 revealed that peroxisomal membrane protein trafficking utilizes canonical ER exit site machinery, connecting peroxisome biogenesis to the secretory pathway.

   Evidence RNAi knockdown and overexpression of Sec16B with fluorescence microscopy and RNAi-resistant rescue in human cells

   PMID:21768384

   Open questions at the time

   • Whether COPII vesicles or a non-vesicular intermediate carries PMPs from ER exit sites was undefined
   • How Sec16B specifically recognizes PEX16 cargo was unknown
   • Other ER exit regulators involved were not identified
5. 2014 High

   Showing that PEX16 mediates ER-to-peroxisome trafficking of PEX3 and PMP34 to pre-existing peroxisomes extended PEX16's role from de novo biogenesis to constitutive peroxisome maintenance, demonstrating continuous ER-to-peroxisome membrane flow.

   Evidence ER-targeted ssPEX3 construct with quantitative live-cell time-lapse microscopy and PEX16 depletion/overexpression

   PMID:25002403

   Open questions at the time

   • Full repertoire of PMPs trafficked in a PEX16-dependent manner was not catalogued
   • The lipid transfer function implied by ER-to-peroxisome flow was not directly measured
   • Structural basis for PEX16-PMP interaction was unknown
6. 2015 High

   Systematic mutagenesis mapped multiple distinct domains in PEX16 responsible for ER-to-peroxisome trafficking versus PMP recruitment, and cross-species complementation with Arabidopsis PEX16 demonstrated conservation of the ER recruitment function across kingdoms.

   Evidence Comprehensive deletion/point mutagenesis of human PEX16 with fluorescence readout; plant PEX16 complementation in human cells

   PMID:25903784

   Open questions at the time

   • Atomic-resolution structure of PEX16 and its interaction surfaces remain undetermined
   • Whether distinct domains act sequentially or in parallel during biogenesis was not resolved
   • Post-translational regulation of PEX16 domain activities was unexplored
7. 2021 Medium

   Discovery that PEX16 depletion activates pexophagy—blocked by ATG5 knockout—revealed that PEX16 modulates peroxisome homeostasis through both biogenesis and degradation arms, establishing a quality-control link.

   Evidence siRNA knockdown of PEX16, ATG5 KO epistasis, chloroquine treatment, p62-ABCD3 co-localization in human cells

   PMID:34360754

   Open questions at the time

   • Single-lab finding; independent confirmation in additional systems needed
   • Mechanism by which PEX16 loss triggers the pexophagy signal was not identified
   • Whether pexophagy is a direct consequence of PMP depletion or a secondary metabolic response was unresolved
8. 2022 High

   CRISPR knockout demonstrated that PEX16 accelerates but is not absolutely required for de novo peroxisomal membrane formation, separating its biogenesis-accelerating activity from PEX3-targeting function and showing that a patient-derived mutant acts as a dominant inhibitor.

   Evidence CRISPR/Cas9 PEX16 KO in three mammalian cell lines, complementation with separation-of-function alleles

   PMID:35437598

   Open questions at the time

   • The PEX16-independent pathway for residual peroxisome formation is molecularly undefined
   • How the patient-derived mutant exerts dominant-negative inhibition at the molecular level was not resolved
   • Relationship between the enlarged peroxisome phenotype and metabolic dysfunction was not characterized

• The structural basis for PEX16's dual functions in PMP recruitment and ER-to-peroxisome trafficking, the identity of the PEX16-independent peroxisome formation pathway, and the molecular signal linking PEX16 loss to pexophagy activation remain to be determined.
• No atomic-resolution structure of PEX16 or its complexes exists
• The alternative peroxisome biogenesis pathway operating without PEX16 is uncharacterized
• Direct PEX16 interactome at the ER membrane has not been comprehensively mapped