Structure of Transmembrane Domain of Lysosome-Associated Membrane Protein Type 2a (LAMP-2A) Reveals Key Features for Substrate Specificity in Chaperone-Mediated Autophagy

J Biol Chem. 2014 Dec 19;289(51):35111-23. doi: 10.1074/jbc.M114.609446. Epub 2014 Oct 22.

Abstract

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.

Keywords: Autophagy; CMA; Chaperone; DPC; LAMP-2; Micelles; Nuclear Magnetic Resonance (NMR); Protein Structure; Transport.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Amino Acid Sequence
  • Autophagy*
  • HSC70 Heat-Shock Proteins / metabolism
  • Humans
  • Kinetics
  • Lysosomal-Associated Membrane Protein 2 / chemistry*
  • Lysosomal-Associated Membrane Protein 2 / genetics
  • Lysosomal-Associated Membrane Protein 2 / metabolism*
  • Magnetic Resonance Spectroscopy / methods
  • Micelles
  • Models, Molecular
  • Molecular Chaperones / metabolism*
  • Molecular Sequence Data
  • Phosphorylcholine / analogs & derivatives
  • Phosphorylcholine / chemistry
  • Protein Binding
  • Protein Multimerization
  • Protein Structure, Secondary
  • Protein Structure, Tertiary*
  • Substrate Specificity

Substances

  • HSC70 Heat-Shock Proteins
  • LAMP2 protein, human
  • Lysosomal-Associated Membrane Protein 2
  • Micelles
  • Molecular Chaperones
  • Phosphorylcholine
  • dodecylphosphocholine

Associated data

  • PDB/2MOF
  • PDB/2MOM