Atlastin Mediated Endoplasmic Reticulum Network Formation In Hereditary Spastic Paraplegia

The endoplasmic reticulum is an extensive multifunctional membrane bound organelle present in all eukaryotic cells. It houses a wide array of essential processes including protein and lipid synthesis, drug detoxification and regulation of intracellular Ca+2 . This very large organelle is organized into morphologically distinct subdomains, presumably to maximize the efficiency of each of its many functions. Yet the ER is interconnected at hundreds of branchpoints. maintaining both luminal and membrane continuity. Despite its complex structure, the ER undergoes continuous membrane remodeling, which may enable it to adopt to environmental changes. Due to their extreme polarity and the long distances that need to be traversed by cellular constituents, neurons may rely more heavily than other cell types on the proper structure, function and dynamics of organelles such as the ER. In support of this idea, a number of neurological disorders are linked to mutations in genes whose products are proposed to structure the ER. In particular, mutations in the neuronal isoform of atlastin (ATL), a conserved dynamin-related GTPase implicated in homotypic ER membrane fusion and ER network formation, cause a motor neurological disorder called Hereditary Spastic Paraplegia (HSP). Determining the role of ATLs in ER morphology has obvious implications in the context of the neurodegeneration seen in Hereditary Spastic Paraplegia patients. To this end, in my thesis I worked on three projects. One focused on testing the hypothesis that disease mutations cause HSP because they disrupt neuronal ATL-1’s fusion-dependent ER structuring function. Using a cell-based assay for ATL-mediated ER network formation, I showed that neuronal ATL-1 can fully restore a branched ER network in HeLa cells depleted of endogenous ATL, and yet surprisingly, not all the disease mutations disrupt ER morphology. Furthermore, at least two disease variants, including that most commonly identified in patients, displayed wild type levels of activity in all assays, including a biochemical assay for membrane fusion. The second project tested the role of an N-terminal extension of ATL-1 that is highly conserved across vertebrate species. My results indicated that this extension was dispensable for ER structuring at least in non-neuronal cells. Therefore, the significant conservation observed within this region may reflect a regulatory role specific to neurons, an idea that remains to be tested. Lastly, I collaborated with James McNew and his group to investigate the precise role of the cytoplasmic C-terminal tail of ATL. Together we showed that the C-terminal tail is important for both the fusion and ER network formation functions of ATL. And yet in the context of less stable lipid bilayers, the requirement for the C-terminal tail during fusion was alleviated. Altogether, my findings reveal a discrepancy with the hypothesis that disease mutations disrupt ER morphology and highlight a gap in the understanding of the cause of ATL-1 linked SPG3A. The apparent lack of a requirement for a highly conserved N-terminal extension, as well as residues implicated in HSP, is surprising. It suggests that the ER in neurons might rely on a neuron specific factor that binds and regulates the fusion. Alternatively, ATL-1 may mediate an additional (non-ER fusion) function specific to neurons. Overall, my investigation reveals that there is more to be understood in terms of precise role (s) and regulation of ATL a well as the basis of SPG3A pathogenesis.</p

Identification of discrete sites in Yip1A necessary for regulation of endoplasmic reticulum structure.

<p>The endoplasmic reticulum (ER) of specialized cells can undergo dramatic changes in structural organization, including formation of concentric whorls. We previously reported that depletion of Yip1A, an integral membrane protein conserved between yeast and mammals, caused ER whorl formation reminiscent of that seen in specialized cells. Yip1A and its yeast homologue Yip1p cycle between the ER and early Golgi, have been implicated in a number of distinct trafficking steps, and interact with a conserved set of binding partners including Yif1p/Yif1A and the Ypt1/Ypt31 Rab GTPases. Here, we carried out a mutational analysis of Yip1A to obtain insight into how it regulates ER whorl formation. Most of the Yip1A cytoplasmic domain was dispensable, whereas the transmembrane (TM) domain, especially residues within predicted TM helices 3 and 4, were sensitive to mutagenesis. Comprehensive analysis revealed two discrete functionally required determinants. One was E95 and flanking residues L92 and L96 within the cytoplasmic domain; the other was K146 and nearby residue V152 within the TM domain. Notably, the identified determinants correspond closely to two sites previously found to be essential for yeast viability (E76 and K130 in Yip1p corresponding to E95 and K146 in Yip1A, respectively). In contrast, a third site (E89) also essential for yeast viability (E70 in Yip1p) was dispensable for regulation of whorl formation. Earlier work showed that E76 (E95) was dispensable for binding Yif1p or Ypt1p/Ypt31p, whereas E70 (E89) was required. Collectively, these findings suggest that the ability of Yip1A to bind its established binding partners may be uncoupled from its ability to control ER whorl formation. In support, Yif1A knockdown did not cause ER whorl formation. Thus Yip1A may use the sites identified herein to interact with a novel binding partner to regulate ER membrane organization.</p

Identification of Discrete Sites in Yip1A Necessary for Regulation of Endoplasmic Reticulum Structure

<div><p>The endoplasmic reticulum (ER) of specialized cells can undergo dramatic changes in structural organization, including formation of concentric whorls. We previously reported that depletion of Yip1A, an integral membrane protein conserved between yeast and mammals, caused ER whorl formation reminiscent of that seen in specialized cells. Yip1A and its yeast homologue Yip1p cycle between the ER and early Golgi, have been implicated in a number of distinct trafficking steps, and interact with a conserved set of binding partners including Yif1p/Yif1A and the Ypt1/Ypt31 Rab GTPases. Here, we carried out a mutational analysis of Yip1A to obtain insight into how it regulates ER whorl formation. Most of the Yip1A cytoplasmic domain was dispensable, whereas the transmembrane (TM) domain, especially residues within predicted TM helices 3 and 4, were sensitive to mutagenesis. Comprehensive analysis revealed two discrete functionally required determinants. One was E95 and flanking residues L92 and L96 within the cytoplasmic domain; the other was K146 and nearby residue V152 within the TM domain. Notably, the identified determinants correspond closely to two sites previously found to be essential for yeast viability (E76 and K130 in Yip1p corresponding to E95 and K146 in Yip1A, respectively). In contrast, a third site (E89) also essential for yeast viability (E70 in Yip1p) was dispensable for regulation of whorl formation. Earlier work showed that E76 (E95) was dispensable for binding Yif1p or Ypt1p/Ypt31p, whereas E70 (E89) was required. Collectively, these findings suggest that the ability of Yip1A to bind its established binding partners may be uncoupled from its ability to control ER whorl formation. In support, Yif1A knockdown did not cause ER whorl formation. Thus Yip1A may use the sites identified herein to interact with a novel binding partner to regulate ER membrane organization.</p> </div

Regions of the Yip1A TM domain required for ER structuring.

<p>(A) Quantification of rescue in cells that were co-transfected with Yip1A siRNA and mutated HA-Yip1A constructs. Data were from 3 independent experiments (>100 cells per experiment), ±SD. Red bars indicate regions that were nonfunctional when mutated to Ala/Leu. (B) A schematic representation of the predicted topology of Yip1A. The results from (A) are represented on the schematic. Residues highlighted in blue were functional and red were nonfunctional when stretches of amino acids were replaced with Ala/Leu. Residues highlighted in yellow were nonfunctional when individual amino acids were replaced with Ala/Leu. Precise substitutions are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054413#pone.0054413.s002" target="_blank">Table S1</a>.</p

Two residues within the Yip1A TM domain are essential for the ER structuring function of Yip1A.

<p>(A) Quantification of cells that were co-transfected with the indicated HA-Yip1A mutated constructs and Yip1A siRNA. Data were from 3 independent experiments (>100 cells per experiment), ±SD. Yellow bars indicate mutations that resulted in a partial rescue. (B, C) Cells co-transfected with Yip1A siRNA and HA-Yip1A K146E and V152L single or double mutant variant constructs were fixed after 72 h and co-stained with HA (B) and calnexin (C) antibodies. Double asterisks indicate cells expressing the double mutant variant that exhibited ER whorls. Scale bar, 10 μm. (D) Quantification of the efficiency of rescue for (B) and (C) from three independent experiments (>100 cells per experiment) ±SD. Single asterisk, p≤0.02 and double asterisk, p<0.0001.</p

Both the cytoplasmic and TM domains of Yip1A are required to regulate ER whorl formation.

<p>HeLa cells were co-transfected with Yip1A siRNA and either a negative control myc-Sec61β construct (not shown), a wild-type HA-Yip1A rescue construct (A–C), a chimeric construct (HA-Yip1AN/Sec61βTM) with the N-terminus of Yip1A fused to the TM helix of Sec61β (D–F), or a Yip1A truncation construct (HA-Yip1A Δ1–118) lacking the entire cytoplasmic domain (G–I). Cells were fixed 72 h after transfection and co-stained with antibodies against HA (A, D, G) or Myc (not shown) and calnexin (B, E, H). Single asterisks mark cells expressing the indicated construct that did not exhibit ER whorls; whereas double asterisks mark expressing cells that did exhibit ER whorls. Scale bar, 10 μm. The constructs are schematized (C, F, I). (J) The normalized efficiency of rescue by each variant was quantified as detailed in Materials and Methods. Data were from 3 independent experiments (>100 cells per experiment), ±SD.</p

Only a few highly conserved residues in the Yip1A cytoplasmic domain are required for function.

<p>Cells co-transfected with Yip1A siRNA and HA-Yip1A Δ1-83 were fixed after 72 h and co-stained with antibodies against HA (A) and calnexin (B). The asterisk (A and B) marks an expressing cell that did not exhibit ER whorls. Scale bar, 10 μm. (C) Quantification of the efficiency of rescue by HA-Yip1A Δ1–83, HA-Yip1A Δ1–118 and the negative control Myc-Sec61β. Data were from 3 independent experiments (>100 cells per experiment), ±SD. (D) An alignment of the cytoplasmic domains of human Yip1A with yeast Yip1p. Residues 83–118 are bracketed, with the highly conserved block highlighted in bold. (E) Bolded residues (in D) were mutated as indicated and tested for rescue. Data from 3 independent experiments (>100 cells per experiment) ±SD are quantified. Yellow bars indicate residues that were partially functional when mutated to alanine. Red bars indicate those same residues showing a more significant loss of function when mutated to a charged residue. Single asterisk, p-value <0.001; double asterisk, p<0.05 (Student's t-test). The open circle and purple bar indicate the previously identified nonfunctional variant E95K <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054413#pone.0054413-Dykstra1" target="_blank">[10]</a>.</p

The Atlastin C-terminal Tail is an Amphipathic Helix that Perturbs Bilayer Structure during Endoplasmic Reticulum Homotypic Fusion

Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum (ER). The large GTPase Atlastin has recently been shown to drive ER membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We have found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel alpha helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion