Localization and targeting of SCG10 to the trans‐Golgi apparatus and growth cone vesicles

Abstract

SCG10 is a membrane-associated, microtubule-destabilizing protein of neuronal growth cones. Using immunoelectron microscopy, we show that in the developing cortex of mice, SCG10 is specifically localized to the trans face Golgi complex and apparently associated with vesicular structures in putative growth cones. Consistent with this, subcellular fractionation of rat forebrain extracts demonstrates that the protein is enriched in the fractions containing the Golgi apparatus and growth cone particles. In isolated growth cone particles, SCG10 was found to be particularly concentrated in the growth cone vesicle fraction. To evaluate the molecular determinants of the specific targeting of SCG10 to growth cones, we have transfected PC12 cells and primary neurons in culture with mutant and fusion cDNA constructs. Deletion of the amino-terminal domain or mutations within this domain that prevented palmitoylation at cysteines 22 and 24 abolished Golgi localization as well as growth cone targeting, suggesting that palmitoylation of the amino-terminal domain is a necessary signal for Golgi sorting and possibly transport of SCG10 to growth cones. Fusion proteins consisting of the amino-terminal domain of SCG10 and the cytosolic proteins stathmin or glutathione-S-transferase colocalized with a Golgi marker, alpha-mannosidase II, and accumulated in growth cones of both axons and dendrites. These results reveal a novel axonal/dendritic growth cone targeting sequence that involves palmitoylation.

Two separate motifs cooperate to target stathmin-related proteins to the Golgi complex

“…In order to identify essential motifs involved in this specific targeting, we examined the subcellular distribution of various subdomains derived from domain A of SCG10 fused with GFP. We show that the Golgi localization of SCG10 results from the cooperation of two motifs: a membrane-anchoring palmitoylation motif and a of growth cones (Stein et al, 1988;Di Paolo et al, 1997a;Gavet et al, 1998;Lutjens et al, 2000;Gavet et al, 2002). SCG10 was also shown to associate with lipid rafts (Maekawa et al, 2001).…”

“…Stathmin-related proteins are peripheral membrane-bound proteins localized to the Golgi complex and on vesicular structures present in the cell body, along neurites, and concentrated within the growth cones of neurons (Stein et al, 1988; Di Paolo et al, 1997a;Lutjens et al, 2000;Gavet et al, 2002). Domain A, common to all stathmin family proteins other than stathmin, was shown here to be sufficient to target GFP to the Golgi complex and vesicles in primary hippocampal neurons, but also in HeLa and MDCK cells, in agreement with previous studies (Di Paolo et al, 1997b;Lutjens et al, 2000).…”

“…More generally we provide new information on essential mechanisms of functional protein subcellular targeting. (Stein et al, 1988; Di Paolo et al, 1997a;Gavet et al, 1998;Lutjens et al, 2000;Gavet et al, 2002). SCG10 was also shown to associate with lipid rafts (Maekawa et al, 2001).…”

“…By contrast, domain A, which is the most Nterminal, is conserved in all stathmin-related proteins (60-70% identity) (Stein et al, 1988;Ozon et al, 1997;Ozon et al, 1998) and has been identified as a key element to target SCG10 to the Golgi complex (Di Paolo et al, 1997b). Indeed, it was shown that not only the deletion of domain A in SCG10 redistributed the protein to the cytosol (Di Paolo et al, 1997b), but also that the addition of this same domain to heterologous proteins was able to target the fusion protein to the Golgi complex in COS-7 cells and in neurons (Di Paolo et al, 1997b;Lutjens et al, 2000). Two palmitoylation sites have been identified in domain A of SCG10, but their mutation did not result in the complete solubilization of the protein (Di Paolo et al., 1997b), suggesting that other signals must be involved in the subcellular localization of stathmin-related proteins to the Golgi complex.…”

“…By characterizing the subcellular localization mediated by various subdomains derived from domain A of SCG10 in fusion with GFP, we delineated and characterized two targeting motifs acting in a cooperative fashion, with membrane-anchoring and Golgi-specifying activities respectively. Altogether, our results open new perspectives concerning the mechanisms by which stathmin family proteins are targeted to their functional compartments and, more generally, concerning specific determinants for Golgi targeting of peripheral membrane proteins.Stathmin-related proteins are peripheral membrane-bound proteins localized to the Golgi complex and on vesicular structures present in the cell body, along neurites, and concentrated within the growth cones of neurons (Stein et al, 1988; Di Paolo et al, 1997a;Lutjens et al, 2000;Gavet et al, 2002). Domain A, common to all stathmin family proteins other than stathmin, was shown here to be sufficient to target GFP to the Golgi complex and vesicles in primary hippocampal neurons, but also in HeLa and MDCK cells, in agreement with previous studies (Di Paolo et al, 1997b;Lutjens et al, 2000).…”