Substrates of N-end rule ubiquitylation and methods for measuring the ubiquitylation of these substrates

A method for producing a conjugate comprising

• (a) at least one
• (i) ubiquitin or
• (ii) ubiquitin derivatized with another molecule, which is employed for purification or visualization;
• and
• (b) a protein selected from the group consisting of aprataxin, SLP, HMG17, PinX1, CIR, HMGN3, HSPC144, Cullin 3, CDC6, tau, and fragments and variants thereof;
• comprising:
• A) forming a mixture comprising a vector containing a clone coding for said protein, or said fragment or variant thereof, an in vitro transcription/translation system, an N-rule ubiquitylation system and, optionally, a proteasome inhibitor;
• and
• B) incubating said mixture to allow production of said conjugate;
• wherein (i) said variants comprise polypeptides of at least 50 amino acids having at least 90% sequence identity to sequences within their corresponding proteins and (ii) said fragments comprise polypeptides of at least 50 amino acids having at least 90% identity to a sequence fragment of said protein;
• and said conjugate is formed via N-end rule ubiquitylation of a polypeptide comprising a destabilizing N-terminal residue and an internal Lys residue.

A composition comprising a conjugate comprised of

• (a) at least one
• (i) ubiquitin or
• (ii) ubiquitin derivatized with another molecule, which is employed for purification or visualization;
• and
• (b) a protein selected from the group consisting of aprataxin, SLP, HMG17, PinX1, CIR, HMGN3, HSPC144, Cullin 3, CDC6, and fragments and variants thereof;
• wherein (i) said variants comprise polypeptides of at least 50 amino acids having at least 90% sequence identity to sequences within their corresponding proteins and (ii) said fragments comprise polypeptides of at least 50 amino acids having at least 90% identity to a sequence fragment of said protein;
• said conjugate is formed via N-end rule ubiquitylation of a polypeptide comprising a destabilizing N-terminal residue plus an internal Lys residue, and said conjugate is immobilized on a support and/or linked to a label.

An isolated conjugate comprising

• (a) at least one
• (i) ubiquitin or
• (ii) ubiquitin derivatized with another molecule, which is employed for purification or visualization;
• and
• (b) a recombinant protein selected from the group consisting of tau, and fragments and variants thereof;
• wherein (i) said variants comprise polypeptides of at least 50 amino acids having at least 90% sequence identity to sequences within their corresponding proteins and (ii) said fragments comprise polypeptides of at least 50 amino acids having at least 90% identity to a sequence fragment of said protein;
• and said conjugate is formed via N-end rule ubiquitylation of a polypeptide comprising a destabilizing N-terminal residue plus an internal Lys residue.

A composition comprising a conjugate comprised of

• (a) at least one
• (i) ubiquitin or
• (ii) ubiquitin derivatized with another molecule, which is employed for purification or visualization;
• and
• (b) a protein selected from the group consisting of tau, and fragments and variants thereof;
• wherein (i) said variants comprise polypeptides of at least 50 amino acids having at least 90% sequence identity to sequences within tau and (ii) said fragments comprise polypeptides of at least 50 amino acids having at least 90% identity to a sequence fragment of said protein;
• and said conjugate is formed via N-end rule ubiquitylation of a polypeptide comprising a destabilizing N-terminal residue plus an internal Lys residue, and said conjugate is immobilized on a support and/or linked to a label.

An isolated conjugate comprising

• (a) at least one
• (i) ubiquitin or
• (ii) ubiquitin derivatized with another molecule, which is employed for purification or visualization;
• and
• (b) a protein;
• said conjugate made by a process comprising:
• A) forming a mixture comprising
• (i) a vector containing a clone coding for said protein, wherein said protein is selected from the group consisting of aprataxin, SLP, HMG17, PinX1, CIR, HMGN3, HSPC144, Cullin 3, CDC6, tau, and fragments and variants thereof;
• and (i) said variants comprise polypeptides of at least 50 amino acids having at least 90% sequence identity to sequences within their corresponding proteins and (ii) said fragments comprise polypeptides of at least 50 amino acids having at least 90% identity to a sequence fragment of said protein;
• and said fragment or variant thereof comprises a destabilizing N-terminal residue and an internal Lys residue;
• (ii) an in vitro transcription/translation system;
• (iii) an N-rule ubiquitylation system;
• and
• (iv) optionally, a proteasome inhibitor;
• and
• B) incubating said mixture to produce said conjugate.