Hereditary spastic paraplegia proteins REEP1, spastin, and atlastin-1 coordinate microtubule interactions with the tubular ER network
Seong H. Park, Peng-Peng Zhu, Rell L. Parker, Craig Blackstone
Seong H. Park, Peng-Peng Zhu, Rell L. Parker, Craig Blackstone
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Research Article Cell biology

Hereditary spastic paraplegia proteins REEP1, spastin, and atlastin-1 coordinate microtubule interactions with the tubular ER network

Abstract

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 tubular ER 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.

Authors

Seong H. Park, Peng-Peng Zhu, Rell L. Parker, Craig Blackstone

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Figure 8

REEP1 interacts preferentially with the M1 isoform of spastin.

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REEP1 interacts preferentially with the M1 isoform of spastin. (A) A sch...
(A) A schematic diagram showing the domain organization of spastin isoforms generated through use of 2 different translation start codons. MIT, present in microtubule-interacting and transport proteins. (B) REEP1 (green) was coexpressed with Myc-tagged M87 (top; red) or M1 spastin (bottom; red) and visualized using confocal microscopy. Scale bars: 10 μm. (C) Alkaline extraction. Myc-tagged M1 spastin but not M87 spastin is present exclusively in the pellet fraction after alkaline extraction, as revealed by immunoblotting for Myc-epitope. MW standards (in kDa) are indicated throughout. (D) Detergent phase partitioning. Membranes from M1 and M87 spastin-expressing cells were partitioned with Triton X-114 (TX-114). Input membranes as well as aqueous (A) and detergent (D) phases were immunoblotted. Partitioning of the soluble protein Grp78 is shown for comparison. (E) Protease protection. Proteinase K was added to intact microsomes from Myc-tagged M1 spastin-expressing cells, with or without Triton X-100, and aliquots were immunoblotted for M1 spastin (Myc-epitope), Grp78, and calnexin. (F) M1 spastins Y52N and G15N create consensus N-linked glycosylation sites but are not glycosylated. WT and the indicated mutant M1 spastin proteins were expressed in COS7 cells and immunoblotted for Myc. (G) Model for REEP1 membrane topology. (H) M1 spastin and REEP1 coimmunoprecipitate. Cells were cotransfected with REEP1 and either Myc-tagged M1 or M87 spastin, then immunoprecipitated with anti-REEP1 antibodies and immunoblotted with anti–Myc-epitope antibodies. The control IgG IP lane used extracts from cells coexpressing REEP1 and M1 spastin.