QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 May 2005
doi: 10.1242/jcs.02349

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Charbaut, E. Articles by Sobel, A. Search for Related Content PubMed PubMed Citation Articles by Charbaut, E. Articles by Sobel, A.

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

Elodie Charbaut^1,*, Stéphanie Chauvin^1,*, Hervé Enslen², Sophie Zamaroczy¹ and André Sobel^1,

¹ INSERM, U706, Neurosignalisation Moléculaire et Cellulaire, Institut du Fer à Moulin, 17 rue du Fer à Moulin, and Université Pierre et Marie Curie (UPMC), 4 place Jussieu, Paris, 75005 France
² INSERM, U536, Transduction du Signal et Plasticité dans le Système Nerveux, Institut du Fer à Moulin, 17, rue du Fer à Moulin, and UPMC, 4 place Jussieu, Paris, 75005 France

View larger version (30K): [in a new window]   Fig. 1. Domains and constructs investigated in this study. (A) Stathmin family proteins: except for stathmin, each protein possesses, in addition to its stathmin-like domain, an N-terminal extension including the conserved domain A (domain A, dark grey; domains A' and A'', symbols). (B) Sequence alignment of domains A from SCG10, SCLIP and RB3/RB3'/RB3'': conserved residues are highlighted in grey, and the two palmitoylated cysteines in black. The delineation of the three regions n (grey), m (black) and c (white) was driven by the sequence conservation pattern. (C) A first series of GFP fusion proteins was constructed with the domain A from SCG10 or with various combinations of its derived subdomains (n+m, m+c, m, n); an additional pair of constructs encoding the palmitoylated N-terminal domain of GAP-43 (hatched) fused or not to region n (lower two constructs) was also engineered to further investigate the mechanism of action of this region.

View larger version (47K): [in a new window]   Fig. 2. The distribution of A-GFP is similar to that of endogenous SCG10 in primary hippocampal neurons. (A) After 5 days in culture, primary hippocampal neurons were transfected with domain A of SCG10 fused to GFP (A-GFP). The fusion protein and the endogenous SCG10 were detected using monoclonal anti-GFP or polyclonal anti-SCG10 antibodies, respectively. Left panel is a low-magnification view of a transfected neuron showing the localization of A-GFP at a perinuclear compartment and along all processes up to their growth cone tips (asterisks). The three images to the right show a high magnification detail of a neuron double-labelled for A-GFP and endogenous SCG10, with the corresponding overlay image showing the colocalization of A-GFP and SCG10 in the perinuclear, Golgi-like region. All images are projections of confocal stacks. (B) Domains A of SCG10, SCLIP and RB3 were fused to GFP and transfected into HeLa cells. In all cases, GFP fluorescence showed a perinuclear as well as vesicular distribution, similar to the subcellular localization described for the corresponding stathmin-related proteins (Gavet et al., 1998). Bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 3. Subcellular distribution of GFP fusion proteins containing domain A of SCG10 or derived subdomains in HeLa cells. A-GFP, anm-GFP, am-GFP, amc-GFP were expressed in HeLa cells. After 24 hours, the cells were fixed and labelled with the CTR433 monoclonal Golgi marker, and examined by fluorescence confocal microscopy. GFP fluorescence and CTR433 immunofluorescence images for each fusion protein, with the corresponding overlay image in the right-hand column (GFP, green; CTR433, red). A-GFP (A) is localized to the Golgi complex, as shown by its colocalization with the CTR433 labelling, and on punctate structures, which may correspond to vesicles. am-GFP (am) and amc-GFP (amc) are present at the Golgi complex but also at the plasma membrane, whereas anm-GFP (anm) is restricted to the Golgi and punctate structures, like the full-length domain A construct. All images are projections from confocal stacks. Bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 4. Subcellular distribution of GFP fusion proteins containing either the N-terminal domain A or derived subdomains, or full-length or n-truncated SCG10 cells. MDCK cells were transfected with (A) A-GFP, anm-GFP, am-GFP, amc-GFP and (B) SCG10-GFP or n-SCG10-GFP constructs, fixed after 24 hours and GFP fluorescence was examined by fluorescence confocal microscopy (A) (images are projections from confocal stacks) or (B) conventional fluorescence microscopy. The subcellular distribution of the proteins is very similar to that observed in HeLa cells: A-GFP (A), anm-GFP (anm) and the full-length SCG10-GFP are mainly restricted to the Golgi complex and punctate structures (in the case of SCG10-GFP, the Golgi is disseminated owing to the microtubule-destabilizing activity of the transfected protein), whereas am-GFP (am), amc-GFP (amc) and n-SCG10-GFP are also present at the plasma membrane. Bar, 10 µm.

View larger version (32K): [in a new window]   Fig. 5. Region n of domain A displays a Golgi-specifying activity when appended to the palmitoylation signal of GAP-43. GFP fusion proteins containing either region n, domain A, the palmitoylated N-terminal domain of GAP-43, or a combination of region n and the N-terminal domain of GAP-43 were transfected in HeLa cells. (A) Conventional fluorescence microscopy image for GFP alone as a control and for each construct. (B) Weakly expressing cells were classified according to the relative distribution of the GFP fusion proteins between the Golgi complex and the plasma membrane. The presence of region n appended to the N-terminal domain of GAP-43 resulted in a higher proportion of fusion protein at the Golgi complex, compared to the protein with the N-terminal domain of GAP-43 alone. G>PM, mainly localized to the Golgi complex; GPM, equally distributed between the Golgi complex and the plasma membrane; PM>G, mainly localized to the plasma membrane. Bar, 10 µm.

View larger version (83K): [in a new window]   Fig. 6. Subcellular localization of A-GFP mutated at specific residues within region n. The indicated mutants (see text for details) were expressed in MDCK cells. Twenty-four hours after transfection, cells were fixed and stained with the CTR433 monoclonal Golgi marker. The subcellular localization of the mutants was examined by conventional fluorescence microscopy. For each fusion protein, GFP fluorescence and CTR433 immunofluorescence images are shown, with the corresponding overlay image (GFP, green; CTR433, red) in the right-hand column. When compared to the wild-type A-GFP (top panels), mutation of charged residues within region n (AYKEKMKEL) to either alanine (KKK/AAA; EE/AA) or to conservatively charged amino acids (KKK/RRR; EE/DD) disturbed the subcellular localization of the resulting fusion proteins. In contrast to the wild type, which is only localized to the Golgi and punctate structures, these mutants were distributed both at the Golgi complex and at the plasma membrane, as clearly observed at cell junctions. Bar, 10 µm.