Protrudin regulates endoplasmic reticulum morphology and function associated with the pathogenesis of hereditary spastic paraplegia

Abstract

Protrudin is a membrane protein that regulates polarized vesicular trafficking in neurons. The protrudin gene (ZFYVE27) is mutated in a subset of individuals with hereditary spastic paraplegia (HSP), and protrudin is therefore also referred to as spastic paraplegia (SPG) 33. We have now generated mice that express a transgene for dual epitope-tagged protrudin under control of a neuron-specific promoter, and we have subjected highly purified protrudin-containing complexes isolated from the brain of these mice to proteomics analysis to identify proteins that associate with protrudin. Protrudin was found to interact with other HSP-related proteins including myelin proteolipid protein 1 (SPG2), atlastin-1 (SPG3A), REEP1 (SPG31), REEP5 (similar to REEP1), Kif5A (SPG10), Kif5B, Kif5C, and reticulon 1, 3, and 4 (similar to reticulon 2, SPG12). Membrane topology analysis indicated that one of three hydrophobic segments of protrudin forms a hydrophobic hairpin domain similar to those of other SPG proteins. Protrudin was found to localize predominantly to the tubular endoplasmic reticulum (ER), and forced expression of protrudin promoted the formation and stabilization of the tubular ER network. The protrudin(G191V) mutant, which has been identified in a subset of HSP patients, manifested an increased intracellular stability, and cells expressing this mutant showed an increased susceptibility to ER stress. Our results thus suggest that protrudin contributes to the regulation of ER morphology and function, and that its deregulation by mutation is a causative defect in HSP.

Keywords: Endoplasmic Reticulum (ER); Endoplasmic Reticulum Stress; Membrane Structure; Neurodegenerative Diseases; Proteomics.

FIGURE 1.

Identification of protrudin-associated proteins with a proteomics approach. A, schematic workflow for identification of protrudin-associated proteins by proteomics analysis. An extract prepared from the brain of protrudin transgenic mice was subjected to dual affinity purification with anti-FLAG and Ni-NTA-agarose. The isolated protrudin complexes were subjected to SDS-PAGE, slices of the resulting gel were exposed to trypsin, and the generated peptides were analyzed by LC-MS/MS. The mass and partial amino acid sequence data were simultaneously compared with protein and nucleotide sequence databases for protein identification. B, subcellular localization of protrudin-associated proteins identified by LC-MS/MS analysis. C, proportion of proteins encoded by genes responsible for the indicated diseases in OMIM that were identified in protrudin complexes. D, proteins directly implicated in or potentially related to HSP identified in A. The amount of each protein was estimated semiquantitatively on the basis of the normalized identification frequency (IF). The proteins were ranked according to the average of the scores from two independent experiments (Exp. 1 and Exp. 2).

FIGURE 2.

Protrudin interacts with atlastin-1 and REEP family members. A–C, extracts of HEK293T cells transiently transfected with expression vectors for FLAG-tagged protrudin and HA epitope-tagged forms of atlastin-1, REEP5, or REEP1 were subjected to immunoprecipitation (IP) with anti-FLAG. VAP-A and VAP-A(ΔTM) were studied as positive and negative controls, respectively, for interaction with protrudin. The resulting precipitates, as well as a portion (1% of the input for immunoprecipitation) of the cell extracts, were subjected to immunoblot (IB) analysis with anti-HA, anti-FLAG, and anti-HSP90 (loading control). D–F, HeLa cells expressing FLAG-tagged protrudin and HA epitope-tagged forms of atlastin-1, REEP5, or REEP1 were fixed and processed for immunofluorescence analysis with anti-FLAG (red) and anti-HA (green). Merged images in which nuclei are stained with Hoechst 33258 (blue) are also shown. The boxed areas in the upper panels are shown at higher magnification in the lower panels. Scale bars, 50 μm. G, domain organization of human protrudin and structure of deletion mutants thereof. RBD, Rab binding domain; HP1 to HP3, hydrophobic domains; CC, coiled-coil domain. H and I, extracts of HEK293T cells expressing full-length (FL) protrudin or its mutants shown in G (fused at their NH₂ termini to the FLAG tag) together with HA-atlastin-1 (H) or HA-REEP5 (I) were subjected to immunoprecipitation with anti-HA, and the resulting precipitates, as well as a portion (1% of the input for immunoprecipitation) of the cell extracts, were subjected to immunoblot analysis with anti-FLAG, anti-HA, and anti-HSP90.

FIGURE 3.

Protrudin is an integral membrane protein. A, cytosolic and membrane fractions were isolated from the brain of WT (+/+) or protrudin-deficient (−/−) mice, and equal amounts of protein from each fraction were subjected to immunoblot analysis with anti-protrudin, anti-HSP90, and anti-FKBP38. HSP90 and FKBP38 were examined as controls for cytosolic and membrane proteins, respectively. B, homogenates of HEK293T cells expressing protrudin tagged at its NH₂ and COOH termini with HA and Myc epitopes, respectively, were subjected to extraction with or without Na₂CO₃ followed by centrifugation to isolate supernatant (S) and pellet (P) fractions. Equal amounts of protein from each fraction were subjected to immunoblot analysis with anti-HA, anti-Myc, anti-GM130, and anti-calnexin. GM130 and calnexin were examined as controls for peripheral and integral membrane proteins, respectively.

FIGURE 4.

Analysis of the membrane topology of protrudin. A, schematic representation of calnexin as well as full-length (FL) and mutant forms of mouse protrudin. The transmembrane (TM) domain of calnexin as well as the hydrophobic regions (HP1 to HP3) of protrudin are indicated. NH₂- or COOH-terminal epitopes recognized by anti-calnexin (calnexin-N and calnexin-C, respectively) are denoted by black bars. Full-length and mutant forms of protrudin were tagged at their NH₂ and COOH termini with HA and Myc epitopes, respectively, as indicated. B, microsomes prepared from HEK293T cells expressing full-length HA-protrudin-Myc were incubated in the absence or presence of proteinase K and Triton X-100 and then subjected to immunoblot analysis with anti-HA, anti-Myc, anti-calnexin-N, or anti-calnexin-C, as indicated. C, microsomes prepared from HEK293T cells expressing HA-protrudin-(1–188)-Myc were analyzed as in B. D, schematic representation of the topology of HA epitope-tagged protrudin mutants. E, HeLa cells were incubated with digitonin to permeabilize the plasma membrane or with Triton X-100 to permeabilize all cell membranes. They were then treated with or without mPEG and subjected to immunoblot analysis with anti-calnexin. F, cysteine residues of protrudin-(1–228) were mutated to alanine, or sequences GGCGG or GGECEGG were inserted before residue 88, as indicated in D. HeLa cells expressing the various HA epitope-tagged protrudin mutants were then analyzed as in E with the exception that immunoblot analysis was performed with anti-HA and anti-calnexin.

FIGURE 5.

Forced expression of protrudin promotes ER network formation. A, COS-7 cells expressing EGFP-tagged Climp63 and FLAG-tagged human protrudin were fixed and processed for immunofluorescence analysis with anti-FLAG (red) and confocal microscopy. The fluorescence of EGFP was monitored directly. Merged images are also shown. The boxed regions of the upper panels are shown at higher magnification in the lower panels. Scale bar, 10 μm. B, COS-7 cells expressing HA epitope-tagged REEP5 and FLAG-tagged human protrudin were fixed and processed for confocal immunofluorescence analysis with anti-HA (green) and anti-FLAG (red). Scale bar, 50 μm. C, percentage colocalization of FLAG-protrudin with EGFP-Climp63 or HA-REEP5 as determined in A and B and as measured with Pearson's correlation coefficient in a square area of 2000 μm². Data are mean ± S.D. from four or five cells. D, COS-7 cells expressing FLAG-tagged human protrudin were fixed and processed for confocal immunofluorescence analysis with anti-Climp63 (green) and anti-FLAG (red). Scale bar, 10 μm. E, COS-7 cells transfected with an expression vector for FLAG-tagged human protrudin (or with the corresponding empty vector, Mock) were fixed and processed for confocal immunofluorescence analysis with anti-calreticulin (green) and anti-FLAG (red). Scale bars, 10 μm. F, percentage colocalization of FLAG-protrudin with Climp63 or calreticulin as determined in D and E and as measured with Pearson's correlation coefficient in a square area of 2000 μm². Data are mean ± S.D. for three cells. G, density of three-way junctions of the ER for cells examined as in E. Data are mean ± S.D. for five cells. A.U., arbitrary units. *, p < 0.05 (Student's t test). H, HeLa cells transfected with protrudin or control siRNAs were subjected to immunoblot analysis with anti-protrudin and anti-HSP90. The arrowhead and asterisk indicate specific and nonspecific bands, respectively. I, HeLa cells transfected with protrudin or control siRNAs were subsequently transfected with an expression vector for FLAG-tagged human protrudin (or with the corresponding empty vector, Mock) before fixation and processing for confocal immunofluorescence analysis with anti-calreticulin (green) and anti-FLAG (red). Left panels are higher magnification views of the boxed areas. Scale bar, 10 μm. J, density of three-way junctions of the ER for cells examined as in I. Data are mean ± S.D. for 7 to 10 cells. *, p < 0.05 (one-way analysis of variance followed by Tukey's test). K, COS-7 cells transfected with expression vectors for tdTomato-tagged Sec61β and FLAG-tagged human protrudin (or with the corresponding empty vector, Mock) were treated (or not) with 100 μm nocodazole for 60 min to induce microtubule depolymerization. The cells were then fixed and processed for immunofluorescence analysis with anti-α-tubulin (green). The fluorescence of tdTomato was monitored directly. Merged images are also shown. The boxed areas in the upper panels are shown at higher magnification in the lower panels. Scale bars, 50 μm. The density of three-way junctions of the ER was also measured. Data are mean ± S.D. for three to seven cells. *, p < 0.05 (Student's t test).

FIGURE 6.

An HSP-associated mutant of protrudin associates with atlastin-1 and REEP family members. A–C, extracts of HEK293T cells transiently transfected with expression vectors for FLAG-tagged WT or G191V mutant forms of human protrudin as well as for HA epitope-tagged forms of atlastin-1, REEP5, or REEP1 were subjected to immunoprecipitation with anti-FLAG. VAP-A(ΔTM) was studied as a negative control for interaction with protrudin. The resulting precipitates, as well as a portion (1% of the input for immunoprecipitation) of the cell extracts, were subjected to immunoblot analysis with anti-HA, anti-FLAG, and anti-HSP90 (loading control). D, COS-7 cells expressing FLAG-tagged WT or G191V mutant forms of protrudin were fixed and processed for confocal immunofluorescence analysis with anti-calreticulin (green) and anti-FLAG (red). The boxed regions of the upper panels are shown at higher magnification in the lower panels. Scale bars, 10 μm. The percentage colocalization of FLAG-protrudin(WT) or FLAG-protrudin(G191V) with calreticulin was measured with Pearson's correlation coefficient in a square area of 2000 μm². Data are mean ± S.D. for four cells. E, COS-7 cells expressing tdTomato-tagged Sec61β and either FLAG-tagged protrudin(WT) or FLAG-protrudin(G191V) were treated (or not) with 100 μm nocodazole for 60 min. The cells were then fixed and processed for immunofluorescence analysis with anti-α-tubulin (green). The fluorescence of tdTomato was monitored directly. Scale bars, 50 μm. The density of three-way junctions of the ER was also measured. Data are mean ± S.D. for four or five cells.

FIGURE 7.

Expression of an HSP-associated mutant of protrudin induces ER stress. A–C, RT and real-time PCR analysis of BiP mRNA in Neuro2A cells infected with retroviruses encoding WT or G191V mutant forms of human protrudin and exposed to 5 μg/ml of tunicamycin (A), 1 μm thapsigargin (B), or 5 mm DTT (C) for 8 h. Data are expressed relative to the corresponding normalized value for cells infected with the empty retrovirus (Mock) and exposed to vehicle, and are mean ± S.D. from four to six independent experiments. *, p < 0.05 (one-way analysis of variance followed by Tukey's test). D, activity of an ER stress response element reporter plasmid relative to that of the reference plasmid pRL-TK in Neuro2A cells expressing WT or G191V mutant forms of protrudin. Data are mean ± S.D. from six independent experiments. *, p < 0.05 (one-way analysis of variance followed by Tukey's test). E, Neuro2A cells infected with retroviruses for HA epitope-tagged WT or G191V mutant forms of protrudin were incubated in the absence or presence of tunicamycin (2 μg/ml) for 7 h and then subjected to RT-PCR analysis of XBP1 mRNA and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA (loading control). Open and filled arrowheads indicate bands corresponding to unspliced (uXBP1) and spliced (sXBP1) forms of XBP1 mRNA, respectively. The asterisk indicates a nonspecific band. The sXBP1/uXBP1 band intensity ratio was measured. F, Neuro2A cells infected with retroviruses for HA epitope-tagged WT or G191V mutant forms of protrudin were incubated with cycloheximide (CHX, 10 μg/ml) for the indicated times, lysed, and subjected to immunoblot analysis with anti-HA, anti-cyclin D1 (positive control), and anti-HSP90 (loading control). The intensity of the HA-protrudin and cyclin D1 bands was measured. Data are mean ± S.D. for four independent experiments. G, Neuro2A cells infected with retroviruses for HA epitope-tagged WT or G191V mutant forms of protrudin were transfected for 48 h with a vector for FLAG-tagged NHK, incubated with cycloheximide (10 μg/ml) for the indicated times, lysed, and subjected to immunoblot analysis with anti-FLAG, anti-HA, anti-cyclin D1, and anti-HSP90. The intensity of the NHK-FLAG and cyclin D1 bands was measured. H, Neuro2A cells infected with retroviruses for HA epitope-tagged WT or G191V mutant forms of protrudin were incubated with cycloheximide (10 μg/ml) in the absence or presence of MG132 (10 μm) for the indicated times, lysed, and subjected to immunoblot analysis with anti-HA, anti-cyclin D1, and anti-HSP90. I, Neuro2A cells infected with retroviruses for HA epitope-tagged forms of WT or G191V mutant forms of protrudin were exposed to 1 μm thapsigargin for 8 h, lysed, and subjected to gel filtration chromatography with a running buffer containing 1% CHAPS. The resulting fractions were subjected to immunoblot analysis with anti-HA and anti-calnexin (negative control), and the intensity of the HA-protrudin and calnexin bands was measured. Similar results were obtained in two independent experiments.