AFC1 and RCE1: isoprenylated CAAX processing enzymes

Abstract

Two genes which encode polypeptides that mediate post-prenylation processing steps in CAAX polypeptides such as Ras are provided. The two genes (AFC1 and RCE1) encode polypeptides that mediate the removal of the AAX tripeptide from the CAAX polypeptide following prenylation. The genes and encoded polypeptides provide assays for testing compounds for an effect on post-prenylation processing steps. A heat shock assay for assessing Ras activity is also provided.

What is claimed is:  
     
         1 . A recombinant expression vector comprising a heterologous promoter operably linked to an expressed polynucleotide which naturally encodes an Afc1 polypeptide, wherein said polypeptide mediates the proteolytic removal of an AAX tripeptide from a prenylated CAAX protein.  
     
     
         2 . A vector according to  claim 1 , wherein the polynucleotide comprises SEQ ID NO:1.  
     
     
         3 . A vector according to  claim 1 , wherein the polypeptide comprises SEQ ID NO:2.  
     
     
         4 . An isolated polynucleotide comprising SEQ ID NO:6 hybridized to an Afc1 transcript.  
     
     
         5 . A recombinant expression vector comprising a promoter operably linked to an expressed polynucleotide which naturally encodes an Rce1 polypeptide, wherein said polypeptide mediates the proteolytic removal of an AAX tripeptide from a prenylated CAAX protein.  
     
     
         6 . A vector according to  claim 5 , wherein the polynucleotide comprises SEQ ID NO:3.  
     
     
         7 . A vector according to  claim 5 , wherein the polypeptide comprises SEQ ID NO:4.  
     
     
         8 . An isolated polynucleotide comprising SEQ ID NO:5 hybridized to an Rce1 transcript.  
     
     
         9 . A recombinant cell transduced with the vector of  claim 1 .  
     
     
         10 . A recombinant cell transduced with the polynucleotide of  claim 4 .  
     
     
         11 . A recombinant cell transduced with the vector of  claim 5 .  
     
     
         12 . A recombinant cell transduced with the polynucleotide of  claim 8 .  
     
     
         13 . A method for making a polynucleotide according to  claim 4 , the method comprising the step of hybridizing a polynucleotide comprising SEQ ID NO:6 with an Afc1 transcript to form a polynucleotide according to  claim 4 .  
     
     
         14 . A method for making a polynucleotide according to  claim 8 , the method comprising the step of hybridizing a polynucleotide comprising SEQ ID NO:5 with an RCE1 transcript to form a polynucleotide according to  claim 8 .  
     
     
         15 . A method of identifying a compound which inhibits the proteolytic removal of an AAX tripeptide of a CAAX protein in a cell, the method comprising steps: 
 contacting a sample comprising a recombinant cell according to  claim 9 , or lysate thereof with a test compound; and    measuring activity or expression of the Afc1p or Rce1p expressed by the cell, wherein compound-dependent inhibition of the activity or expression indicates that the compound inhibits the proteolytic removal of the AAX tripeptide.    
     
     
         16 . A method of identifying a compound which inhibits the proteolytic removal of an AAX tripeptide of a CAAX protein in a cell, the method comprising steps: 
 contacting a sample comprising a recombinant cell according to  claim 10 , or lysate thereof with a test compound; and    measuring activity or expression of the Afc1p or Rce1p expressed by the cell, wherein compound-dependent inhibition of the activity or expression indicates that the compound inhibits the proteolytic removal of the AAX tripeptide.    
     
     
         17 . A method of identifying a compound which inhibits the proteolytic removal of an AAX tripeptide of a CAAX protein in a cell, the method comprising steps: 
 contacting a sample comprising a recombinant cell according to  claim 11 , or lysate thereof with a test compound; and    measuring activity or expression of the Afc1p or Rce1p expressed by the cell, wherein compound-dependent inhibition of the activity or expression indicates that the compound inhibits the proteolytic removal of the AAX tripeptide.    
     
     
         18 . A method of identifying a compound which inhibits the proteolytic removal of an AAX tripeptide of a CAAX protein in a cell, the method comprising steps: 
 contacting a sample comprising a recombinant cell according to  claim 12 , or lysate thereof with a test compound; and    measuring activity or expression of the Afc1p or Rce1p expressed by the cell, wherein compound-dependent inhibition of the activity or expression indicates that the compound inhibits the proteolytic removal of the AAX tripeptide.    
     
     
         19 . A method of identifying a compound which inhibits Rce1p activity or Afc1p activity, the method comprising steps: 
 expressing from a polynucleotide according to  claim 4  an Afc1 or Rce1 polypeptide;    isolating the polypeptide;    contacting a test compound to a sample comprising the isolated polypeptide; and    measuring an activity selected from the group consisting of Afc1p activity, Rce1p activity, Afc1p expression, and Rce1p expression,    wherein compound-dependent inhibition of the activity indicates that the compound inhibits Rce1p activity or Afc1p activity.    
     
     
         20 . A method of identifying a compound which inhibits Rce1p activity or Afc1p activity, the method comprising steps: 
 expressing from a polynucleotide according to  claim 8  an Afc1 or Rce1 polypeptide;    isolating the polypeptide;    contacting a test compound to a sample comprising the isolated polypeptide; and    measuring an activity selected from the group consisting of Afc1p activity, Rce1p activity, Afc1p expression, and Rce1p expression,    wherein compound-dependent inhibition of the activity indicates that the compound inhibits Rce1p activity or Afc1p activity.