We have previously shown that myosin VI is present at the Golgi complex (Buss et al., 1998
) and is involved not only in the steady-state organization of the Golgi complex but also in post-Golgi membrane traffic (Warner et al., 2003
). To investigate the precise roles that myosin VI plays in these processes we identified optineurin, a peripheral Golgi protein as a binding partner for myosin VI. This interaction found in a yeast two-hybrid screen was confirmed using a range of different protein–protein interaction studies. Myosin VI and optineurin colocalize at the Golgi complex and in vesicular structures close to the plasma membrane. The loss of optineurin not only leads to Golgi fragmentation and a reduction in exocytosis, but we also show that optineurin is essential for targeting myosin VI to the Golgi complex. Most interestingly optineurin plays a role in colocalizing myosin VI and Rab8, a member of the Rab family of G-proteins, which are important regulators of membrane traffic events.
Optineurin binds directly to the sequence RRL (residues 1107–1109) in the globular tail of myosin VI. This binding site is distinct from the Dab2-binding site and is independent of the large insert. The binding of optineurin to myosin VI is regulated by phosphorylation at the TINT (1088–1091) site in the globular tail region. Our results suggest that a PAK phosphorylates myosin VI not only in the motor domain (Buss et al., 1998
) but also in the globular tail domain at threonine 1088 and 1091, thus coordinating the regulation of motor function with cargo binding in the tail domain.
In the absence of optineurin the Golgi complex is fragmented. The ribbon structure of interconnected stacks of membrane cisternae is broken up and the disconnected Golgi stacks are dispersed throughout the cytoplasm. The overall appearance of the fragmented Golgi stacks, however, is similar to the appearance of the Golgi in control cells and no gross vesiculation was observed at the EM level. Golgi resident proteins such as TGN46 and GM130 were still present in the Golgi fragments. These morphological changes are very similar to those observed when microtubules are disrupted by drugs such as nocodazole (Rogalski and Singer, 1984
), suggesting that optineurin might be involved in linking Golgi membranes directly or indirectly to microtubules around the microtubule organizing center (MTOC). One of the optineurin-binding partners is huntingtin, known to interact with HAP1 (Huntingtin-associated protein; Li et al., 1995
), which binds directly to the dynactin subunit p150glued
(Engelender et al., 1997
; Li et al., 1998a
). The dynactin complex is involved in linking the minus end directed microtubule motor protein dynein to membrane vesicles (Gill et al., 1991
). It has been proposed that huntingtin and HAP1 act as scaffolding proteins regulating the interaction between the dynein–dynactin complex and cargo such as Golgi membranes (Harjes and Wanker, 2003
). The absence or inactivation of cytoplasmic dynein leads to fragmentation and spreading of the Golgi complex into the cytoplasm (a phenotype similar to that observed after the loss of optineurin; Harada et al., 1998
); thus it is possible that optineurin via the huntingtin–HAP1 complex may be involved in targeting dynein to Golgi membranes (). Recent results (Gauthier et al., 2004
) indicate a further role for the huntingtin–HAP1 complex in the axonal transport of vesicles containing brain-derived neurotrophic factor along microtubules and highlight the importance of huntingtin-binding partners in the progression of the disease.
Figure 9. A cartoon illustrating the possible interaction of optineurin with motor protein complexes at the Golgi complex. Optineurin may play a central role in coordinating actin-based and microtubule-based motor function for maintaining Golgi morphology. The (more ...)
The loss of optineurin not only disrupts the structure of the Golgi complex, but also dramatically reduces secretion of VSV-G to the plasma membrane. Reduced transport is not due to Golgi fragmentation because Golgi stacks dispersed by depolymerization of microtubules remain fully functional with only a slight reduction in protein secretion (Van De Moortele et al., 1993
). Reduced exocytosis of VSV-G in the optineurin knockdown cells is a very similar phenotype to that observed in the myosin VI knockout mouse, where secretion of alkaline phosphatase is also significantly reduced (Warner et al., 2003
). Secretion in these cells lacking myosin VI is not restored by mutant myosin VI, which is unable to bind to optineurin, indicating that a functional complex between myosin VI and optineurin is required for constitutive exocytosis.
Rab8 binds to optineurin and plays an important role in polarized membrane transport both from the Golgi complex to the basolateral membrane in polarized epithelial cells and to dendrites in neurons (Huber et al., 1993a
). Rab8 is present in the same intracellular compartments as myosin VI and in this paper we show that myosin VI and Rab8 colocalize in the perinuclear region around the Golgi complex and both are present on the same vesicles underneath the plasma membrane. In addition Rab8 has been localized to the recycling endosome, a compartment shown to be involved in post-Golgi membrane trafficking to the plasma membrane (Ang et al., 2003
). However, at present we don't know whether some myosin VI is also associated with the fraction of Rab8 present in the recycling endosome. On the other hand because Rab8 may interact via optineurin with myosin VI it suggests a possible pathway whereby TGN-derived vesicles are transported short distances along actin filaments by myosin VI, before a kinesin motor picks up the cargo for long distance transport along microtubules. Similarly, close to the plasma membrane the Rab8–optineurin–myosin VI complex might be involved in presenting the secretory vesicle to the docking site, thus tethering the vesicle close to the plasma membrane before fusion. Overexpression of Rab8-Q67L leads to the formation of long tubular extensions from the Golgi complex and it recruits endogenous myosin VI onto these tubular structures. These results indicate that Rab8 serves as a membrane receptor for myosin VI, recruiting this myosin motor onto a specific intracellular membrane compartment via optineurin. Similarly, it was shown recently that Rab27a recruits myosin Va onto melanosomes via the linker molecule melanophillin (Seabra and Coudrier, 2004
Optineurin is the first myosin VI–binding partner identified at the Golgi complex and may play an adaptor or linker role. The recent demonstration that myosin VI can exist as a nonprocessive monomer (Lister et al., 2004
) and also possibly as a processive dimer (Rock et al., 2001
; Nishikawa et al., 2002
) may provide a mechanism for the distinct dual roles for myosin VI in structural maintenance and vesicle transport action away from the Golgi complex. We therefore suggest that optineurin may serve two major “linker” functions for myosin VI in the Golgi complex (): First, optineurin together with huntingtin links via the minus end directed motor dynein to the microtubule network and also by directly interacting with myosin VI, is linked to the actin cytoskeleton, thus it may play a role in coordinating microtubule-based and actin-based motor activity around the Golgi complex. Support for such a role is provided by our previous observation that in Snell's waltzer mice the absence of myosin VI causes a reduction in the size of the Golgi complex (Warner et al., 2003
), which would be due to the dynein motor complex pulling the Golgi membranes in toward the MTOC resulting in a smaller more compact Golgi complex. Therefore, in the absence of myosin VI the balance of motor proteins is disturbed and dynein wins the “tug of war”. Second, optineurin might link myosin VI to Rab8, a regulatory protein known to be involved in sorting molecules in the exocytic pathway at the TGN and in membrane fusion at the plasma membrane. The linker function of optineurin may be temporally regulated, for example by phosphorylation of individual proteins, including myosin VI and optineurin (Schwamborn et al., 2000
In addition, because optineurin is obviously a dimer with multiple leucine zippers it may induce monomeric myosin VI to dimerize for processive movement of vesicles from the TGN to the plasma membrane.
Our work provides new information on the intracellular functions of optineurin, which will help elucidate how mutations in the optineurin gene lead to primary open angle glaucoma (Rezaie et al., 2002
). Previous reports, that reduced secretion of the glycoprotein myocilin leads to primary open angle glaucoma (Joe et al., 2003
), support our results that optineurin plays a major role in Golgi morphology and exocytosis.