Nuclear import is required for the pro-apoptotic function of the Golgi protein p115.
During apoptosis the Golgi apparatus undergoes irreversible fragmentation. In part, this results from caspase-mediated cleavage of several high molecular weight coiled-coil proteins, termed golgins. These include GM130, golgin 160, and the Golgi vesicle tethering protein p115, whose caspase cleavage generates a C-terminal fragment (CTF) of 205 residues. Here we demonstrate that early during apoptosis, following the rapid cleavage of p115, endogenous CTF translocated to the cell nucleus and its nuclear import was required to enhance the apoptotic response. Expression of a series of deletion constructs identified a putative alpha-helical region of 26 amino acids, whose expression alone was sufficient to induce apoptosis; deletion of these 26 residues from the CTF diminished its proapoptotic activity. This region contains several potential SUMOylation sites and co-expression of SUMO together with the SUMO ligase, UBC9, resulted in SUMOylation of the p115 CTF. Significantly, when cells were treated with drugs that induce apoptosis, SUMOylation enhanced the efficiency of p115 cleavage and the kinetics of apoptosis. A construct in which a nuclear export signal was fused to the N terminus of p115 CTF accumulated in the cytoplasm and surprisingly, its expression did not induce apoptosis. In contrast, treatment of cells expressing this chimera with the antibiotic leptomycin induced its translocation into the nucleus and resulted in the concomitant induction of apoptosis. These results demonstrate that nuclear import of the p115 CTF is required for it to stimulate the apoptotic response and suggest that its mode of action is confined to the nucleus.
Mukherjee
Shaeri
S
Department of Developmental, Albert Einstein College of Medicine, Bronx, New York 10461, USA. smukherj@aecom.yu.edu
Shields
Dennis
D
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D017433:Protein Structure, Secondary / Q000502:physiology; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D000935:Antifungal Agents; D001324:Autoantigens; C108388:GOLGA3 protein, human; D000077614:Golgi Matrix Proteins; C097741:Golgin subfamily A member 2; D008565:Membrane Proteins; D025842:SUMO-1 Protein; D033921:Vesicular Transport Proteins; C091580:vesicular transport factor p115; D044763:Ubiquitin-Conjugating Enzymes; D020169:Caspases; D000098843:Ubiquitin-Conjugating Enzyme UBC9