Cellular Localization, Oligomerization, and Membrane Association of the Hereditary Spastic Paraplegia 3A (SPG3A) Protein Atlastin

Abstract

Hereditary spastic paraplegias comprise a group of clinically heterogeneous syndromes characterized by lower extremity spasticity and weakness, with distal axonal degeneration in the long ascending and descending tracts of the spinal cord. The early onset hereditary spastic paraplegia SPG3A is caused by mutations in the atlastin/human guanylate-binding protein-3 gene (renamed here atlastin-1), which codes for a 64-kDa member of the dynamin/Mx/guanylate-binding protein superfamily of large GTPases. The atlastin-1 protein is localized predominantly in brain, where it is enriched in pyramidal neurons in the cerebral cortex and hippocampus. In cultured cortical neurons, atlastin-1 colocalized most prominently with markers of the Golgi apparatus, and immunogold electron microscopy revealed a predominant localization of atlastin-1 to the cis-Golgi. Yeast two-hybrid analyses and co-immunoprecipitation studies demonstrated that atlastin-1 can selfassociate, and gel-exclusion chromatography and chemical cross-linking studies indicated that atlastin-1 exists as an oligomer in vivo, most likely a tetramer. Membrane fractionation and protease protection assays revealed that atlastin-1 is an integral membrane protein with two predicted transmembrane domains; both the N-terminal GTP-binding and C-terminal domains are exposed to the cytoplasm. Together, these findings indicate that the SPG3A protein atlastin-1 is a multimeric integral membrane GTPase that may be involved in Golgi membrane dynamics or vesicle trafficking.

Intra- and Intermolecular Domain Interactions of the C-terminal GTPase Effector Domain of the Multimeric Dynamin-like GTPase Drp1

“…Immunocytochemistry-COS-7 cells were grown on glass coverslips and transfected as described previously (29,30). In some experiments, 100 nM Mitotracker Red CMXRos (Molecular Probes, Eugene, OR) was added (37°C, 30 min) before fixation.…”

“…N-terminal c-Myc (EQKLISEEDL, single-letter amino acid code) or HA epitope tags (YPYDVPDYA) were engineered into pGW1 after an initiator methionine residue where indicated (29,30). For yeast twohybrid tests, Drp1 deletion constructs were produced by PCR amplification using PfuTurbo TM polymerase, incorporating EcoRI restriction sites for subcloning in-frame into pGAD10 or pBHA (BD Biosciences, Palo Alto, CA).…”

“…DNA Constructs-Complementary DNAs for full-length Homo sapiens Drp1 (GenBank TM accession number AB006965) or C-and N-terminal deletions were generated by PCR using PfuTurbo TM DNA polymerase (Stratagene, La Jolla, CA) with EcoRI ends added and cloned into the EcoRI site of the mammalian expression vector pGW1 (29,30). N-terminal c-Myc (EQKLISEEDL, single-letter amino acid code) or HA epitope tags (YPYDVPDYA) were engineered into pGW1 after an initiator methionine residue where indicated (29,30).…”

“…Yeast Two-hybrid Tests-Yeast two-hybrid assays were performed as described previously using the L40 yeast strain, with selection by HIS3 and ␤-galactosidase induction (29,30). Interactions were semi-quantified based on growth on ϪHis/Leu/Trp plates supplemented with 5 mM 3-amino-1,2,4-triazole and time for colonies to turn blue in X-gal filter lift assays at room temperature, with similar results obtained in at least three experiments (29).…”

Charakterisierung molekularer Mechanismen der anti-angiogenen Aktivität von GBP-1 in Endothelzellen

“…Bis heute konnten sieben humane (GBP-1 bis GBP-7) und zehn murine GBPMitglieder mit einem Molekulargewicht zwischen 65 und 71 kDa identifiziert werden (Degrandi et al, 2007;Olszewski et al, 2006;Tripal et al, 2007). Neben der GBP-Subfamilie besteht die Familie der großen GTPasen aus weiteren Mitgliedern, wie den Atlastinen (Zhu et al, 2003), den Mx-Proteinen (Aebi et al, 1989) und den Dynaminen (Diatloff-Zito et al, 1995). Über die Expression und Funktion der verschiedenen humanen GBPs ist bisher sehr wenig bekannt.…”