The COOH terminus of GATE-16, an intra-Golgi transport modulator, is cleaved by the human cysteine protease HsApg4A.
Docking of a vesicle at the appropriate target membrane involves an interaction between integral membrane proteins located on the vesicle (v-SNAREs) and those located on the target membrane (t-SNAREs). GATE-16 (Golgi-associated ATPase enhancer of 16 kDa) was shown to modulate the activity of SNAREs in the Golgi apparatus and is therefore an essential component of intra-Golgi transport and post-mitotic Golgi re-assembly. GATE-16 contains a ubiquitin fold subdomain, which is terminated at the carboxyl end by an additional amino acid after a conserved glycine residue. In the present study we tested whether the COOH terminus of GATE-16 undergoes post-translational cleavage by a protease which exposes the glycine 116 residue. We describe the isolation and characterization of HsApg4A as a human protease of GATE-16. We show that GATE-16 undergoes COOH-terminal cleavage both in vivo and in vitro, only when the conserved glycine 116 is present. We then utilize an in vitro assay to show that pure HsApg4A is sufficient to cleave GATE-16. The characterization of this protease may give new insights into the mechanism of action of GATE-16 and its other family members.
Scherz-Shouval
Ruth
R
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Sagiv
Yuval
Y
Shorer
Hagai
H
Elazar
Zvulun
Z
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D001692:Biological Transport; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D050356:Lipid Metabolism; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; C498698:GABARAPL2 protein, human; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C474025:ATG4A protein, human; D003546:Cysteine Endopeptidases; D005998:Glycine