The carbohydrate-binding domain of overexpressed STBD1 is important for its stability and protein-protein interactions

Biosci Rep. 2014 Jul 1;34(4):e00117. doi: 10.1042/BSR20140053.

Abstract

STBD1 (starch-binding domain-containing protein 1) belongs to the CBM20 (family 20 carbohydrate binding module) group of proteins, and is implicated in glycogen metabolism and autophagy. However, very little is known about its regulation or interacting partners. Here, we show that the CBM20 of STBD1 is crucial for its stability and ability to interact with glycogen-associated proteins. Mutation of a conserved tryptophan residue (W293) in this domain abolished the ability of STBD1 to bind to the carbohydrate amylose. Compared with the WT (wild-type) protein, this mutant exhibited rapid degradation that was rescued upon inhibition of the proteasome. Furthermore, STBD1 undergoes ubiquitination when expressed in COS cells, and requires the N-terminus for this process. In contrast, inhibition of autophagy did not significantly affect protein stability. In overexpression experiments, we discovered that STBD1 interacts with several glycogen-associated proteins, such as GS (glycogen synthase), GDE (glycogen debranching enzyme) and Laforin. Importantly, the W293 mutant of STBD1 was unable to do so, suggesting an additional role for the CBM20 domain in protein-protein interactions. In HepG2 hepatoma cells, overexpressed STBD1 could associate with endogenous GS. This binding increased during glycogenolysis, suggesting that glycogen is not required to bridge this interaction. Taken together, our results have uncovered new insights into the regulation and binding partners of STBD1.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy / genetics
  • COS Cells
  • Carbohydrates / genetics*
  • Carcinoma, Hepatocellular / genetics
  • Carcinoma, Hepatocellular / metabolism
  • Cell Line
  • Cell Line, Tumor
  • Chlorocebus aethiops
  • Glycogen / genetics
  • Glycogen / metabolism
  • Glycogenolysis / genetics
  • HEK293 Cells
  • HeLa Cells
  • Hep G2 Cells
  • Humans
  • Liver Neoplasms / genetics
  • Liver Neoplasms / metabolism
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism*
  • Mutation / genetics
  • Proteasome Endopeptidase Complex / genetics
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Interaction Domains and Motifs / genetics*
  • Tryptophan / genetics
  • Tryptophan / metabolism
  • Ubiquitination / genetics

Substances

  • Carbohydrates
  • Membrane Proteins
  • Tryptophan
  • Glycogen
  • Proteasome Endopeptidase Complex