Targeting of SCG10 to the Area of the Golgi Complex Is Mediated by Its NH2-terminal Region

Gilbert Di Paolo,Robert Lutjens,Véronique Pellier,Stephen A Stimpson,Marie-Hélène Beuchat,Stefan Catsicas,Gabriele Grenningloh

doi:10.1074/jbc.272.8.5175

Gilbert Di Paolo, Robert Lutjens + Show 5 more

Open Access

https://doi.org/10.1074/jbc.272.8.5175

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Abstract

SCG10 is a neuronal growth-associated protein that is concentrated in the growth cones of developing neurons. SCG10 shows a high degree of sequence homology to the ubiquitous phosphoprotein stathmin, which has been recently identified as a factor that destabilizes microtubules by increasing their catastrophe rate. Whereas stathmin is a soluble cytosolic protein, SCG10 is membrane-associated, indicating that the protein acts in a distinct subcellular compartment. Identifying the precise intracellular distribution of SCG10 as well as the mechanisms responsible for its specific targeting will contribute to elucidating its function. The main structural feature distinguishing the two proteins is that SCG10 contains an NH2-terminal extension of 34 amino acids. In this study, we have examined the intracellular distribution of SCG10 in PC12 cells and in transfected COS-7 cells and the role of the NH2-terminal domain in membrane-binding and intracellular targeting. SCG10 was found to be localized to the Golgi complex region. We show that the NH2-terminal region (residues 1-34) was necessary for membrane targeting and Golgi localization. Fusion proteins consisting of the NH2-terminal 34 amino acids of SCG10 and the related protein stathmin or the unrelated protein, beta-galactosidase, accumulated in the Golgi, demonstrating that this sequence was sufficient for Golgi localization. Biosynthetic labeling of transfected COS-7 cells with [3H]palmitic acid revealed that two cysteine residues contained within the NH2-terminal domain were sites of palmitoylation.

Highlights

Types (Ref. 1; for review see Ref. 6)
We demonstrate that the NH2-terminal region of SCG10 contains a signal required for its targeting to the area of the Golgi complex and sufficient to target stathmin as well as the unrelated protein, ␤-galactosidase, to this subcellular region
To investigate the nature of the punctate structures, we performed double stainings of PC12 cells with two markers of the Golgi complex: ␣-mannosidase II, a glycoprotein enriched in the cis/medial Golgi region [23] and wheat germ agglutinin (WGA), a lectin that binds to N-acetylglucosamine and Nacetylneuraminic acid residues found predominantly in the trans Golgi apparatus/network [24] (Fig. 2, C–F)

Summary

Introduction

Types (Ref. 1; for review see Ref. 6). In cells of the immune system, a role for the control of cell cycle has been proposed [7,8,9]. We have previously found that in PC12 cells, stathmin is required for neuronal differentiation in response to nerve growth factor (NGF)1 [10]. We have defined the intracellular localization of SCG10 in PC12 cells and transfected COS-7 cells as the area of the Golgi complex. We demonstrate that the NH2-terminal region of SCG10 contains a signal required for its targeting to the area of the Golgi complex and sufficient to target stathmin as well as the unrelated protein, ␤-galactosidase, to this subcellular region. The neuronal growth-associated protein SCG10 belongs to the same gene family as stathmin, a ubiquitous phosphoprotein. SCG10 shares 74% amino acid identity with stathmin [5], which has been implicated in signal transduction mechanisms due to its phosphorylation in response to extracellular stimuli regulating proliferation and differentiation in different cell

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