Atlastin-1

[Kenneth Larson]

The ATL1 gene provides instructions for producing a protein called atlastin-1. Atlastin-1 is produced primarily in the brain and spinal cord (central nervous system), particularly in nerve cells (neurons) that extend down the spinal cord (corticospinal tracts). These neurons send electrical signals that lead to voluntary muscle movement. In neurons, this protein is found mainly in the endoplasmic reticulum, which is a structure involved in protein processing and distribution. Atlastin-1 fuses together the network of tubules that make up the structure of the endoplasmic reticulum. Atlastin-1 is also active in compartments called axonal growth cones, which are located at the tip of neurons. The axonal growth cones direct the growth of specialized extensions, called axons, which transmit nerve impulses that signal muscle movement. Within axonal growth cones, atlastin-1 acts during development to help guide the growth of axons.

Approximately 60 mutations in the ATL1 gene have been found to cause spastic paraplegia type 3A. This condition is characterized by muscle stiffness (spasticity) and weakness of the lower limbs (paraplegia), which begin in childhood. Most of the mutations that cause spastic paraplegia type 3A change one protein building block (amino acid) in the atlastin-1 protein. These mutations likely lead to abnormal activity of atlastin-1, which impairs the functioning of neurons, including the distribution of materials within these cells. This lack of functional atlastin-1 protein can also restrict the growth of axons. Within the long neurons of the corticospinal tracts, these problems can lead to cell death. As a result, the neurons are unable to transmit nerve impulses, particularly to other neurons and muscles in the lower extremities. This impaired nerve function leads to the signs and symptoms of spastic paraplegia type 3A.

Mutations in the ATL1 gene have been found to cause a condition called hereditary sensory neuropathy type ID. This condition is characterized by nerve abnormalities in the legs and feet (peripheral neuropathy). Many people with this condition experience prickling or tingling sensations (paresthesias), absent reflexes, weakness, and a reduced ability to feel pain. Affected individuals often get open sores (ulcers) on their feet, and because they cannot feel the pain of these sores, they may not seek immediate treatment. Without treatment, the ulcers can become infected and may require amputation of the surrounding area.

At least four ATL1 gene mutations have been found to cause hereditary sensory neuropathy type ID. These mutations impair nerve cell function and decrease transmission of nerve impulses, similar to the effects of ATL1 gene mutations that cause spastic paraplegia type 3A (described above). It is unclear why some ATL1 gene mutations cause hereditary sensory neuropathy type ID and others cause spastic paraplegia type 3A.

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Hereditary spastic paraplegias (HSPs; SPG1-45) are inherited neurological disorders characterized by lower extremity spastic weakness. More than half of HSP cases result from autosomal dominant mutations in atlastin-1 (also known as SPG3A), receptor expression enhancing protein 1 (REEP1; SPG31), or spastin (SPG4). The atlastin-1 GTPase interacts with spastin, a microtubule-severing ATPase, as well as with the DP1/Yop1p and reticulon families of ER-shaping proteins, and SPG3A caused by atlastin-1 mutations has been linked pathogenically to abnormal ER morphology. Here we investigated SPG31 by analyzing the distribution, interactions, and functions of REEP1. We determined that REEP1 is structurally related to the DP1/Yop1p family of ER-shaping proteins and localizes to the ER in cultured rat cerebral cortical neurons, where it colocalizes with spastin and atlastin-1. Upon overexpression in COS7 cells, REEP1 formed protein complexes with atlastin-1 and spastin within the tubular ER, and these interactions required hydrophobic hairpin domains in each of these proteins. REEP proteins were required for ER network formation in vitro, and REEP1 also bound microtubules and promoted ER alignment along the microtubule cytoskeleton in COS7 cells. A SPG31 mutant REEP1 lacking the C-terminal cytoplasmic region did not interact with microtubules and disrupted the ER network. These data indicate that the HSP proteins atlastin-1, spastin, and REEP1 interact within the tubularER membrane in corticospinal neurons to coordinate ER shaping and microtubule dynamics. Thus, defects in tubular ER shaping and network interactions with the microtubule cytoskeleton seem to be the predominant pathogenic mechanism of HSP.

Atlastin, a member of the dynamin superfamily, is known to catalyse homotypic membrane fusion in the smooth endoplasmic reticulum (ER). Recent studies of atlastin have elucidated key features about its structure and function; however, several mechanistic details, including the catalytic mechanism and GTP hydrolysis-driven conformational changes, are yet to be determined. Here, we present the crystal structures of atlastin-1 bound to GDPAlF(4)(-) and GppNHp, uncovering an intramolecular arginine finger that stimulates GTP hydrolysis when correctly oriented through rearrangements within the G domain. Utilizing Frster Resonance Energy Transfer, we describe nucleotide binding and hydrolysis-driven conformational changes in atlastin and their sequence. Furthermore, we discovered a nucleotide exchange mechanism that is intrinsic to atlastin's N-terminal domains. Our results indicate that the cytoplasmic domain of atlastin acts as a tether and homotypic interactions are timed by GTP binding and hydrolysis. Perturbation of these mechanisms may be implicated in a group of atlastin-associated hereditary neurodegenerative diseases.

RCSB PDB Core Operations are funded by the U.S. National Science Foundation (DBI-2321666), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198.

By positional cloning, Zhao et al. (2001) demonstrated that a form of autosomal dominant hereditary spastic paraplegia with early onset (SPG3A; 182600), before the age of 10 years and usually before the age of 5, is caused by mutation in a GTPase gene. The gene was found to have no homology to genes that cause other forms of hereditary spastic paraplegia. It does show significant homology to guanylate binding protein-1 (GBP1; 600411), a member of the dynamin family of large GTPases. Northern blot analysis of ATL1 expression detected a 2.2-kb transcript primarily in adult and fetal brain. RT-PCR experiments indicated measurable expression in all tissues examined, although expression in adult brain was at least 50-fold higher than in other tissues. Translation of the 2.2-kb cDNA sequence of ATL1 yielded a peptide of 558 amino acids.

By PCR of a cerebral cortex cDNA library, Zhu et al. (2003) cloned ATL1, which they called atlastin-1. The deduced 558-amino acid protein contains GTP-binding motifs in its N-terminal half and 2 transmembrane domains in its C-terminal half. It also has 3 potential N-glycosylation sites. Western blot analysis of transfected COS-7 cells detected atlastin-1 at an apparent molecular mass of about 64 kD. Western blot analysis detected high atlastin-1 expression in human and rat brain homogenates, with much lower expression in several other human tissues, including smooth muscle, adrenal gland, kidney, testis, and lung. Immunohistochemical analysis of rat brain sections detected high expression of atlastin-1 in cortical neurons of lamina V, pyramidal neurons in CA1 and CA3 of the hippocampus, and in amygdala and several thalamic nuclei. Staining was most prominent in the cell soma, with weaker staining of axons and dendrites. Immunogold labeling detected rat atlastin-1 predominantly in the cis-Golgi cisternae. Protease protection assays indicated that the N and C termini of human atlastin-1 were exposed to the cytoplasmic face of the membrane in transfected cells.

Using a yeast 2-hybrid assay, Zhu et al. (2003) determined that atlastin-1 self-associates. Chemical cross-linking experiments indicated that atlastin-1 most likely forms a homotetramer of about 230 kD. Zhu et al. (2003) demonstrated that atlastin-1 has GTPase activity.

Independently, Evans et al. (2006) and Sanderson et al. (2006) demonstrated that the N-terminal domain of spastin (SPAST; 604277) bound directly to the C-terminal cytoplasmic domain of atlastin, suggesting that the 2 gene products interact in a common biologic pathway. Evans et al. (2006) used yeast 2-hybrid analysis and coimmunoprecipitation studies in HeLa cells, and Sanderson et al. (2006) used yeast 2-hybrid analysis of a human fetal brain cDNA library and protein pull-down, coimmunoprecipitation, and colocalization studies in HeLa cells, HEK293T cells, and mouse NSC34 neuronal cells.

In the developing rat brain, Zhu et al. (2006) found that atlastin-1 was expressed not only in the Golgi apparatus and endoplasmic reticulum, but was also enriched in axonal growth cones and growth cone-like varicosities along the axons. Atlastin-1 labeling was prominent on vesicular structures within the growth cones, but not at the plasma membrane and not at synapses. Knockdown of atlastin-1 using shRNA in cultured cortical cells inhibited axonal growth. Overall, the findings suggested that atlastin-1 has diverse functions in neurons, likely acting both in intracellular membrane trafficking as well as in expansion at the axonal growth cone. These functional studies suggested that the early-onset axonopathy observed in SPG3A may result from abnormal development of axons.

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