STOP-like Protein 21 Is a Novel Member of the STOP Family, Revealing a Golgi Localization of STOP Proteins

摘要

Neuronal microtubules are stabilized by two calmodulin-regulated microtubule-associated proteins, E-STOP and N-STOP, which when suppressed in mice induce severe synaptic and behavioral deficits. Here we show that mature neurons also contain a 21-kDa STOP-like protein, SL21, which shares calmodulin-binding and microtubule-stabilizing homology domains with STOP proteins. Accordingly, in different biochemical or cellular assays, SL21 has calmodulin binding and microtubule stabilizing activity. However, in cultured hippocampal neurons, SL21 antibodies principally stain the somatic Golgi and punctate Golgi material in neurites. In cycling cells, transfected SL21 decorates microtubules when expressed at high levels but is otherwise principally visible at the Golgi. The Golgi targeting of SL21 depends on the presence of cysteine residues located within the SL21 N-terminal domain, suggesting that Golgi targeting may require SL21 palmitoylation. Accordingly we find that SL21 is palmitoylated in vivo. N-STOP and E-STOP, which contain the Golgi targeting sequences present in SL21, also display distinct Golgi staining when expressed at low level in cycling cells. Thus neuronal proteins of the STOP family have the capacity to associate with Golgi material, which could be important for STOP synaptic functions. Neuronal microtubules are stabilized by two calmodulin-regulated microtubule-associated proteins, E-STOP and N-STOP, which when suppressed in mice induce severe synaptic and behavioral deficits. Here we show that mature neurons also contain a 21-kDa STOP-like protein, SL21, which shares calmodulin-binding and microtubule-stabilizing homology domains with STOP proteins. Accordingly, in different biochemical or cellular assays, SL21 has calmodulin binding and microtubule stabilizing activity. However, in cultured hippocampal neurons, SL21 antibodies principally stain the somatic Golgi and punctate Golgi material in neurites. In cycling cells, transfected SL21 decorates microtubules when expressed at high levels but is otherwise principally visible at the Golgi. The Golgi targeting of SL21 depends on the presence of cysteine residues located within the SL21 N-terminal domain, suggesting that Golgi targeting may require SL21 palmitoylation. Accordingly we find that SL21 is palmitoylated in vivo. N-STOP and E-STOP, which contain the Golgi targeting sequences present in SL21, also display distinct Golgi staining when expressed at low level in cycling cells. Thus neuronal proteins of the STOP family have the capacity to associate with Golgi material, which could be important for STOP synaptic functions. Neurons contain abundant subpopulations of stable microtubules that resist depolymerizing conditions such as exposure to the cold. This property is due to microtubule association with E-and N-STOP 2The abbreviations used are: STOP, stable tubule only polypeptide; SL21, 21-kDa STOP-like protein; aa, amino acid(s); DIV, days (of differentiation) in vitro; mAb, monoclonal antibody; GST, glutathione S-transferase; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium., two neuronal calmodulin-binding and calmodulin-regulated proteins (1Andrieux A. Salin P.A. Vernet M. Kujala P. Baratier J. Gory-Fauré S. Bosc C. Pointu H. Proietto D. Schweitzer A. Denarier E. Klumperman J. Job D. Genes Dev. 2002; 16: 2350-2364Crossref PubMed Scopus (146) Google Scholar, 2Guillaud L. Bosc C. Fourest-Lieuvin A. Denarier E. Pirollet F. Lafanechère L. Job D. J. Cell Biol. 1998; 142: 167-179Crossref PubMed Scopus (108) Google Scholar, 3Bosc C. Frank R. Denarier E. Ronjat M. Schweitzer A. Wehland J. Job D. J. Biol. Chem. 2001; 276: 30904-30913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). STOP proteins are important for synaptic function and STOP null mice present defects in both short- and long-term synaptic plasticity, associated with severe behavioral and neurotransmitter deficits