Interaction of phosducin and phosducin isoforms with a 26S proteasomal subunit, SUG1

Mol Vis. 1998 Aug 11;4:13.

Abstract

Purpose: Retinal phosducin (Phd) and phosducin-like protein 1 (PhLP1) selectively bind G-protein beta/gamma subunits (Gbetagamma). Our laboratory has recently identified two phosducin-like orphan proteins (PhLOP1 and PhLOP2) that lack the ability to interact with Gbetagamma. In search of potential functional protein partner(s) for these phosducin orphans, we examined their protein-protein interactions using a yeast two-hybrid screen.

Methods: A bovine retina yeast expression cDNA library was screened with the GAL4 DNA binding domain (BD) fusion of PhLOP1. Quantitative analysis of the selected positives with PhLOP1 and other Phd isoforms was assessed by growth and beta-galactosidase activity. Further molecular, biochemical, and immunological detection methods utilizing glutathione S-transferase (GST)-Phd isoform fusion proteins and the potential partner were also performed.

Results: A member of the superfamily of putative ATPases was selected in the yeast two hybrid screen. Further characterization identified a direct interaction of this putative ATPase with PhLOP1, as well as Phd and PhLP1, but not with PhLOP2. A database search verified this ATPase as a bovine orthologue of the yeast SUG1 (ySUG1), a putative transcriptional mediator and a subunit of the 26S proteasome complex. Our experiments reveal that the carboxy-terminus of PhLOP1 is essential for the protein-protein interaction with SUG1, but it alone is not sufficient to mediate SUG1 interaction.

Conclusions: Based on these experimental results, Phd, PhLP1 and PhLOP1 have protein-protein interaction with SUG1. PhLOP1, a truncated amino-terminal splice variant of Phd, is the best candidate for the interaction with SUG1 among the four Phd isoforms studied, which suggests a potential function for PhLOP1.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphatases
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cattle
  • DNA, Complementary / metabolism
  • Eye Proteins / genetics*
  • Eye Proteins / metabolism*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • GTP-Binding Protein Regulators
  • GTP-Binding Proteins / antagonists & inhibitors
  • Gene Library
  • Molecular Sequence Data
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Phosphoproteins / genetics*
  • Phosphoproteins / metabolism*
  • Proteasome Endopeptidase Complex
  • Protein Binding
  • Protein Kinases
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Retina / chemistry
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins*

Substances

  • Carrier Proteins
  • DNA, Complementary
  • Eye Proteins
  • Fungal Proteins
  • GTP-Binding Protein Regulators
  • Nerve Tissue Proteins
  • PDCL protein, human
  • Phosphoproteins
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • phosducin
  • phosducin-like orphan protein-1
  • phosducin-like orphan protein-2
  • Protein Kinases
  • Proteasome Endopeptidase Complex
  • Adenosine Triphosphatases
  • GTP-Binding Proteins
  • RPT6 protein, S cerevisiae