Protrudin binds atlastins and endoplasmic reticulum-shaping proteins and regulates network formation

Abstract

Hereditary spastic paraplegias are inherited neurological disorders characterized by progressive lower-limb spasticity and weakness. Although more than 50 genetic loci are known [spastic gait (SPG)1 to -57], over half of hereditary spastic paraplegia cases are caused by pathogenic mutations in four genes encoding proteins that function in tubular endoplasmic reticulum (ER) network formation: atlastin-1 (SPG3A), spastin (SPG4), reticulon 2 (SPG12), and receptor expression-enhancing protein 1 (SPG31). Here, we show that the SPG33 protein protrudin contains hydrophobic, intramembrane hairpin domains, interacts with tubular ER proteins, and functions in ER morphogenesis by regulating the sheet-to-tubule balance and possibly the density of tubule interconnections. Protrudin also interacts with KIF5 and harbors a Rab-binding domain, a noncanonical FYVE (Fab-1, YGL023, Vps27, and EEA1) domain, and a two phenylalanines in an acidic tract (FFAT) domain and, thus, may also function in the distribution of ER tubules via ER contacts with the plasma membrane or other organelles.

Fig. 1.

Membrane topology of protrudin. (A) Myc-tagged protrudin or atlastin-1 were transfected into HEK293T cells as indicated. Homogenates (Homog) were resolved into soluble and membrane (Memb) fractions, and aliquots were immunoblotted (IB). Calnexin and β-tubulin are markers for membrane and cytosolic fractions, respectively. (B) Membrane fractions from cells expressing HA-protrudin or REEP1-HA were phase partitioned with Triton X-114 and then immunoblotted for HA-epitope or Grp78. (C) Protease protection assays. Microsomes from Myc-protrudin–expressing cells were treated with PK. Aliquots were immunoblotted with Myc-epitope (N terminus of protrudin) or anti-protrudin (C-terminal region of protrudin) antibodies. Calnexin (partly luminal) and Grp78 (completely luminal) were used to monitor proteolysis. A major proteolytic fragment of protrudin is denoted with an arrow. (D) Cells were transfected with wild-type (WT) Myc-protrudin or else Y127N and V149N mutants, and lysates were immunoblotted as shown. (E) Membrane topology model for protrudin. CC, coiled-coil; RBD, Rab-binding domain. Migrations of molecular-mass standards (in kilodaltons) are to the left in B–D.

Fig. 2.

Protrudin localizes to the ER via its intramembrane domains and interacts with ER-shaping proteins. (A) From top to bottom, HeLa cells transiently expressing HA-protrudin were immunostained for endogenous REEP5 or calreticulin (red), along with HA-epitope (green). HeLa cells stably expressing HA-protrudin were immunostained for endogenous REEP5 or CLIMP-63 (green) and HA-epitope (red). Cells expressing Myc–atlastin-3 were coimmunostained for Myc-epitope (green) and endogenous protrudin (red). HeLa cells were costained for endogenous calnexin (green) and endogenous protrudin (red). Insets in the merged images (with DAPI staining) are enlarged to the right. (B) Lysates from cells stably expressing HA-protrudin were immunoblotted as shown. Endo., endogenous. (C, Upper) Schematic diagram of human protrudin and ΔTM deletion constructs. CC, coiled-coil; RBD, Rab-binding domain. (C, Lower) Cells expressing HA-tagged wild-type (WT) protrudin and the indicated deletion mutants were stained for HA-epitope (green) and DAPI (blue). (D) Wild-type and mutant protrudin proteins as in C were expressed in HEK293T cells. Homogenates (Homog) were separated into soluble and membrane (Memb) fractions and immunoblotted. (E) Membrane fractions from cells expressing wild-type and mutant protrudin proteins were phase partitioned with Triton X-114 and then immunoblotted for HA-epitope or Grp78. (F) Endogenous protrudin in HEK293T cells was immunoprecipitated (IP) with anti-protrudin antibodies or control IgG, and precipitates were immunoblotted as shown. ATL, atlastin; KTN, kinectin. (Scale bars: 10 μm.)

Fig. 3.

Protrudin plays a role in forming the tubular ER network. (A) Two different siRNAs against human protrudin were transfected into HeLa cells for 72 h, and cell lysates were immunoblotted. An asterisk (*) indicates a cross-reacting band, and an arrow identifies protrudin. Migrations of molecular-mass standards are to the right. (B) siRNA-transfected HeLa cells were immunostained for CLIMP-63 (green) and REEP5 (red). Insets are enlarged to the right of each image. (Scale bars: 10 μm.) Line-scan plots are shown in Fig. S6A. (C) Numbers of cells with expanded CLIMP-63 signal area were quantified in control and protrudin-depleted conditions (n = 3; >200 cells per experiment). Means ± SD are shown. Paired Student t test: **P < 0.01; ***P < 0.001.

Fig. 4.

Protrudin depletion modulates the ER sheet-to-tubule balance. (A) HeLa cells were transfected with control (siCTL) (n = 33) or protrudin (n = 22) siRNAs for 72 h and then immunostained with CLIMP-63 (green) and α-tubulin (red). All studies were done at the same time, with the same procedures and microscope settings. Pixel areas of CLIMP-63 were normalized to those of α-tubulin using ImageJ tools. Data are presented in arbitrary units. Means ± SEM are shown. Student t test: ***P < 0.001. (Scale bar: 10 μm.) (B) siRNA-transfected HeLa cells were immunogold-labeled for a luminal epitope of CLIMP-63, with visualization by electron microscopy. N, nucleus. Arrowheads denote the plasma membrane. (Scale bars: 500 nm.)