15 Feb 2023
by Richa

Summary- paper 16: The Adaptor Protein-1 μ1B Subunit Expands the Repertoire of Basolateral Sorting Signal Recognition in Epithelial Cells

Xiaoli Guo, Rafael Mattera, Xuefeng Ren, Yu Chen, Claudio Retamal, Alfonso González, Juan S. Bonifacino

Developmental Cell, 2013

Questions/gaps addressed:

  • Epithelial cells are polarized into an apical side that faces the exterior or lumen of body structures and a basolateral side that contacts neighboring cells and the underlying basement membrane. Protein sorting to the basolateral plasma membrane is mediated by signals in their cytosolic tails. AP-1 adaptor plays an important role in basolateral protein sorting.

  • Why do polarized epithelial cells express two isoforms of the μ1 subunit of the AP-1 clathrin adaptor complex: the ubiquitous μ1A and the epithelial-specific μ1B? What are the differences between these isoforms and how do they affect function of their respective AP-1 complexes?

  • μ1A and μ1B are highly homologous (∼80% overall amino acid sequence identity in mammals).

Major hypotheses:

  • Since μ1B is epithelial-specific, hypothesized that μ1B might confer on AP-1 the ability to recognize cargoes that are not efficiently recognized by μ1A.

Key methods:

  • μ1A and μ1B tagged at the C termini with a 10-aa spacer (GSGSGGSGSG) followed by three copies of the hemagglutinin (HA) or Myc epitope. Transient transfection into nonpolarized Madin-Darby canine kidney (MDCK) epithelial cells. Immunostaining and confocal fluorescence microscopy showed localization of both proteins to a juxtanuclear structure characteristic of the TGN/REs, and colocalized with endogenous γ-adaptin, but not early endosomes (EEA1).

  • C-terminal tag was functional based on LDLR trafficking to the basolateral surface. N-terminal tagging does not work well for the µ subunits.

  • Superresolution structured illumination microscopy (SR-SIM), which has a resolution limit of ∼100 nm to look at co-localization of µ1A and µ1B. They co-localize in non-polarized cells. Saw the same thing with TIRF microscopy.

  • MDCK cells grown into polarized monolayers on Transwell filters. Confocal fluorescence microscopy showed localization of endogenous γ-adaptin to a subapical compartment characteristic of the TGN/REs. μ1A-GFP and μ1B-mCherry colocalized to the same subapical compartment.

  • Subcellular fractionation on 40%–60% sucrose gradients to examine the distribution of μ1A-HA and μ1B-Myc. Both cosedimented on 40%–60% sucrose gradients in association with clathrin-coated vesicles (CCVs) containing clathrin and γ-adaptin.

  • Arf1 (regulated by BFA-sensitive GEFs) and Arf6 (regulated by BFA-sensitive and BFA-insensitive GEFs). Dominant-negative Arf1 mutant (Arf1-T31N) displaced both AP-1A and AP-1B complexes from TGN/REs to cytosol, Arf6 mutant (Arf6-T27N) was less efficient.

  • Glutathione S-transferase (GST) pull-down assay to compare the binding of recombinant AP-1A and AP-1B core complexes to different Arfs in vitro. Both AP-1 variants bound to constitutively active forms of class I and II Arfs: Arf1, Arf4, and Arf5 and, to a lesser extent, Arf6.

  • Screened the cytosolic tails of a large number of cargo proteins for interaction with μ1A and μ1B, using a yeast two-hybrid (Y2H).
    • Most tails that tested positive in this assay interacted with both μ1A and μ1B e.g., LAMP1.
    • Preferential binding to μ1A : interleukin-6 receptor α chain [IL6R-α]
    • Preferential binding to μ1B : interleukin-6 receptor β chain [IL6R-β], poliovirus receptor [PVR], low density lipoprotein receptor [LDLR].
      • The LDLR tail contains two basolateral sorting signals: a proximal signal comprising a tyrosine residue (Y828) and an acidic patch (EDE 833-835), and a distal signal comprising two tyrosine residues (Y845 and Y847) and another acidic patch (EED 856-858). All of these elements were required for interaction with μ1B.
  • Pull-down using full-length LDLR tail fused to maltose-binding protein (MBP) and recombinant AP-1A and AP-1B core complexes tagged with GST, in the absence or presence of the constitutively active Arf1 Q71L mutant to test for interactions with the locked or open conformations of the AP-1 core. Pull-down with amylose beads followed by immunoblotting with antibody to GST. LDLR tail bound AP-1B ∼5-fold more avidly than AP-1A. Arf1 Q71L activated binding of both, and binding was dependent on Y845 and Y847 in the LDLR tail.

Major takeaways:

  • the μ1A and μ1B subunit isoforms of AP-1 were previously proposed to specify localization of the complex to different intracellular compartments. Demonstrated that is not the case.

  • Correlation between basolateral sorting and preferential interaction with μ1B for at least three cargo proteins (i.e., IL6R-β, PVR, and LDLR)