Evidence for a Menkes-like protein with a nuclear targeting sequence.

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Menkes disease is a rare genetic disorder affecting copper transport, caused by mutations in the ATP7A gene.
• ATP7A encodes a copper-transporting P-type ATPase essential for cellular copper homeostasis.
• Abridged gene transcripts with novel insertion sequences have been identified in human cell lines.

Purpose of the Study:

• To investigate a specific abridged Menkes disease gene transcript variant (NML 45) containing a novel insertion sequence.
• To determine the functional significance of the insertion sequence within the NML 45 transcript.
• To explore the potential role of this transcript variant in cellular copper transport.

Main Methods:

• Analysis of human cell line extracts for abridged Menkes disease gene transcripts.
• Molecular cloning and expression of a green fluorescent protein (GFP) construct containing the NML 45 transcript variant.
• Transfection of GFP constructs into Chinese hamster ovary cells to assess protein localization.
• Comparison of nuclear localization of NML 45-GFP with constructs lacking the insertion or containing wild-type ATP7A exons.

Main Results:

• A transcript variant, NML 45, was identified, coding for a protein with the first copper-binding domain of ATP7A and a 45-bp insert.
• The 66-nucleotide segment within the insert encodes 22 amino acids with nuclear-localization sequence (NLS) properties.
• GFP constructs with NML 45 localized to the cell nucleus, while constructs without the insert or with wild-type ATP7A exons did not.

Conclusions:

• The 22-residue segment in the NML 45 transcript contains a functional NLS.
• This NLS directs an 11.2-kDa protein with one copper-binding site to the cell nucleus.
• The findings suggest a novel mechanism for copper transport into the nucleus via a chaperone protein encoded by this transcript variant.