REEP4

REEP4 is an endoplasmic reticulum membrane protein that coordinates ER morphogenesis with mitotic progression and nuclear envelope organization (PMID:23911198, PMID:30995177). Together with the closely related REEP3, it binds microtubules through a short positively charged sequence between its two hydrophobic domains and clears ER membranes from metaphase chromosomes; loss of REEP3/4 causes ER to accumulate on mitotic chromatin and produces aberrant intranuclear membranes in daughter cells (PMID:23911198). Distinct from this clearance function, its reticulon homology domain drives the high-curvature ER tubulation characteristic of mitotic ER, with the C-terminus regulating this activity (PMID:30995177). A separate pool of REEP4 is recruited to the inner nuclear membrane by the NPC biogenesis factor ELYS, where it promotes nuclear pore complex assembly, particularly during mitosis (PMID:34874453). Its cytoplasmic domain carries a phosphorylation-dependent 14-3-3-binding site, linking REEP4 to phospho-regulatory signaling (PMID:21725060). In vertebrate development, REEP4 is required to maintain nervous-system and muscle gene expression during early embryogenesis (PMID:19123125).

1. 2009 Medium

   Before any molecular role was assigned, it was unknown whether REEP4 was physiologically required in a whole organism; knockdown established an essential role in early vertebrate development.

   Evidence Antisense morpholino knockdown in Xenopus tropicalis with marker gene in situ analysis

   PMID:19123125

   Open questions at the time

   • No molecular mechanism connecting REEP4 to neural/muscle maintenance
   • Phenotype from a single study with no rescue control reported
2. 2011 Medium

   It was unknown how REEP4 might be regulated; phosphoproteomics identified a phosphorylation-dependent 14-3-3-binding site in its cytoplasmic domain, placing REEP4 under phospho-signaling control.

   Evidence 14-3-3 affinity purification, MS phosphoproteomics, and binding-site validation

   PMID:21725060

   Open questions at the time

   • Functional consequence of 14-3-3 binding undefined
   • Kinase responsible for the phosphorylation not identified
3. 2013 High

   The cellular function of REEP4 was unresolved; a microtubule-membrane linker screen showed REEP3/4 bind microtubules via a positively charged linker and redundantly clear ER from metaphase chromatin, defining a role in mitotic ER positioning and downstream nuclear envelope integrity.

   Evidence Biochemical linker screen, siRNA depletion in HeLa cells, live imaging, EM, and domain mutagenesis

   PMID:23911198

   Open questions at the time

   • Redundancy with REEP3 prevents assigning REEP4-specific contribution
   • How the linker engages microtubules structurally not resolved
4. 2019 High

   It was unclear whether REEP3/4 only repositioned ER or also shaped it; RHD-dependent ER tubulation during mitosis was shown to be a function mechanistically distinct from chromatin clearance.

   Evidence siRNA depletion, live and electron microscopy of ER morphology, and RHD/C-terminus domain dissection

   PMID:30995177

   Open questions at the time

   • Regulatory inputs governing the C-terminus-mediated control not defined
   • Relationship to the 14-3-3 site unexplored
5. 2021 High

   Whether REEP4 acted beyond the cytoplasmic ER was unknown; recruitment to the inner nuclear membrane by ELYS revealed a direct role in nuclear pore complex assembly, especially during mitotic NPC biogenesis.

   Evidence Fluorescence localization, ELYS Co-IP, and siRNA depletion with NPC assembly assays

   PMID:34874453

   Open questions at the time

   • Mechanism by which REEP4 promotes NPC assembly not defined
   • Whether ER-shaping and NPC-assembly pools are functionally separable unclear

• How REEP4's phospho-regulation, microtubule binding, ER-shaping, and ELYS-dependent NPC functions are integrated across the cell cycle remains unresolved.
• No unified model linking 14-3-3 signaling to its mitotic functions
• REEP4-specific roles separate from REEP3 redundancy not established
• No structural model of the protein