Controlling the localization of enzymes within the cell is an important general mechanism for regulating their activity. Posttranslational modifications and conformational changes are known to retarget many cytoplasmic enzymes from one place to another, but dynamic changes in the distribution of enzymes within the ER lumen are less well characterized. We find that the TorA-binding partner LULL1—an ER transmembrane protein—drives redistribution of this lumenal AAA+ ATPase from throughout the ER network specifically into the NE (), where previous in vitro and in vivo studies have suggested a function (Goodchild and Dauer, 2004
; Naismith et al., 2004
; Goodchild et al., 2005
). The idea that regulated distribution between the ER and NE might control TorA's activity toward spatially restricted substrate(s) arose initially from studies showing that so-called “substrate trapped” TorA mutants accumulated in the NE (Gerace, 2004
; Goodchild and Dauer, 2004
; Naismith et al., 2004
). The wild-type enzyme, in contrast, is diffusely distributed throughout the ER except in a few cell types (Giles et al., 2008
) and after certain pharmacological manipulations (Hewett et al., 2003
; Nery et al., 2008
). We hypothesized that additional factor(s), which perhaps are more abundant in cell types where TorA exhibits NE preference (Giles et al., 2008
), were therefore likely to control the targeting and activity of wild-type TorA. Extrapolating between the extremes of LULL1 overexpression (, , and ) and LULL1 silencing () leads us to propose that transient interaction with LULL1 positively regulates the targeting and activity of TorA in the NE. The abundance of LULL1 thus emerges as a critical regulator of TorA activity. Future studies will address whether variations in endogenous LULL1 levels explain previously described cell type–specific differences in TorA distribution (Giles et al., 2008
) and whether there are peaks in the expression or stabilization of LULL1 that correlate with important events in neuronal development or plasticity.
Figure 9. Model of LULL1-dependent TorA function at the NE. LULL1 interacts with the TorA hexamer in the peripheral ER, which promotes an activating change in TorA that requires catalytic residues and association with the membrane. TorA then moves into the NE, (more ...)
LULL1 is a ~70-kDa single-pass transmembrane protein with no defined functional motifs and no known binding partners other than TorA (Goodchild and Dauer, 2005
). Several features suggest that LULL1 may be regulated at both the transcriptional and posttranslational levels, making it an attractive potential regulatory protein. There are significant variations in the level of LULL1 message in different cells and tissues (Goodchild and Dauer, 2005
; Chen et al., 2006
) and over the course of in vitro muscle cell differentiation (Chen et al., 2006
). Further, the extralumenal domain of LULL1 has a preponderance of charged residues and a dearth of hydrophobic residues, both of which are likely to predispose it to rapid and potentially regulated turnover (Fink, 2005
). Consistently, secondary structure prediction algorithms indicate a lack of stably folded structures in this region, and the PESTfind algorithm (http://www.at.embnet.org/toolbox/pestfind
) finds two high-scoring sequences that may correlate with rapid protein turnover (Rechsteiner and Rogers, 1996
). Finally, as a transmembrane protein, LULL1 provides a way to directly or indirectly transmit signals across the ER membrane to TorA.
Using LULL1 overexpression to concentrate TorA in the NE, we were able to uncover molecular and structural changes that are likely to represent TorA's normal activity, including alterations in a subset of NE proteins that participate in NE-bridging LINC complexes ( and ). LINC complexes are formed when INM-localized Sun proteins and ONM-localized nesprin proteins interact within the NE lumen and have recently attracted attention as important connectors between the cytoskeleton, NE membranes, and elements within the nucleus including the lamina and telomeres (Starr, 2009
). Although much remains to be learned about the cell biology of LINC complexes, it is clear that they have important roles in such diverse processes as nuclear anchorage, cell migration, and regulating gene expression (Worman and Gundersen, 2006
; Crisp and Burke, 2008
). Our data showing that some LINC complex proteins—Sun2, nesprin-2G, and nesprin-3—are destabilized by TorA as it accumulates in the NE suggest possible roles for TorA in these same processes. In support of this, a recent study showed that fibroblasts from TorA knockout mice migrate more slowly than controls in a polarized cell migration assay (Nery et al., 2008
Although LINC complex proteins may help recruit TorA to the NE, the subset that TorA displaces (i.e., at least Sun2, nesprin-2G, and nesprin-3) seem unlikely to be directly responsible for retaining it there because TorA remains in the NE even after these proteins are gone. Future work will need to explore possible roles for other NE proteins in this process, perhaps especially remaining LINC complex proteins such as Sun1 and the known TorA-binding partner LAP1.
The mechanism by which LULL1 induces TorA retargeting is an area of ongoing interest. Here, we present several observations about TorA that constrain the possible explanations. Using BN-PAGE () and FRAP (), we established that TorA assembles into a large, membrane-associated oligomer—probably a hexamer—within the ER lumen. The oligomeric state of TorA did not change after interaction with LULL1, and LULL1 remained highly mobile whether or not it was transiently engaging and affecting TorA. These results are the first demonstration that TorA indeed assembles into the kind of oligomer expected of an AAA+ protein and establish that LULL1 changes TorA targeting without affecting its fundamental structure. Our mutagenesis experiments indicate that an N-terminal hydrophobic sequence distinct from TorA's core AAA+ domain (Kock et al., 2006
; Zhu et al., 2008
) is required for this retargeting (). It is therefore attractive to hypothesize that interaction with LULL1 causes a conformational change involving this N-terminal domain, enhancing TorA's affinity for something within the NE. Future work will focus on defining these states and further delineating the mechanism(s) responsible for controlling the transition between the ER-distributed and NE-enriched forms of TorA.
Importantly, we found that DYT1
-associated ΔGAG-TorA is also redirected to the NE by LULL1 ( and ), but once there it is less effective at enacting changes in NE structure and protein composition (). These results suggest a molecular loss-of-function that may correlate with the previously described inability of ΔGAG-TorA to rescue the lethality of TorA knockout in the mouse (Dang et al., 2005
; Goodchild et al., 2005
) and could ultimately contribute to the development of DYT1
dystonia. At a structural level, comparison of TorA's AAA+ domain to that of ClpB or ClpA suggests that the ΔGAG deletion falls in a position that could perturb a helix preceding an ATP-interacting loop known as the sensor-2 motif thereby leading to a loss-of-function (Kock et al., 2006
; Zhu et al., 2008
). The fact that TorA is now established to be an oligomeric enzyme () supports the possibility that mixed oligomers containing wild-type and mutant subunits could turn a loss-of-function mutation into a dominantly inherited trait. Separately, data from other groups have shown that overexpressing ΔGAG-TorA can have toxic effects on the function of the secretory pathway (Hewett et al., 2007
), raising the possibility that a combination of the loss-of-function shown here and gain-of-function shown elsewhere might explain the dominant inheritance of DYT1
dystonia. The discovery that LULL1 regulates the distribution and activity of TorA within the ER and the NE paves the way for future exploration of how changes in its activity correlate with the development of disease.