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Abstract
The Menkes disease ATPase (MNK) is a copper transporter that localizes to the mammalian trans-Golgi network (TGN) and shows substantial co-localization with a ubiquitous TGN resident protein and marker, TGN46. We tested our hypothesis that these two TGN residents and integral membrane proteins are localized to biochemically distinct TGN sub-compartments using constitutively active mutant proteins and drugs that disrupt membrane traffic, lumenal pH and the cellular cytoskeleton. The pH-disrupting agent, monensin, causes MNK to be more diffusely distributed with partial separation of staining patterns for these two TGN residents. Expression of a constitutively active Rho-kinase (ROCK-KIN), which causes formation of juxta-nuclear astral actin arrays, also effects separation of MNK and TGN46 staining patterns. Treatment of ROCK-KIN expressing cells with latrunculin B, an actin-depolymerizing agent, causes complete overlap of MNK and TGN46 staining patterns with concomitant disappearance of polymerized actin. When microtubules are depolymerized in ROCK-KIN expressing cells by nocodazole, both MNK and TGN46 are found in puncate structures throughout the cell. However, a substantial proportion of MNK is still found in a juxta-nuclear location in contrast to TGN46. Actin distribution in these cells reveals that juxta-nuclear MNK is distinct to the astral actin clusters in ROCK-KIN expressing cells where the microtubules were depolymerized. The TGN to cell-surface transport of MNK requires both actin and microtubule networks, whilst the constitutive trafficking of proteins is independent of actin. Taken together, our findings indicate that at least two TGN sub-domains are regulated by separate cytoskeletal dynamics involving actin and tubulin.
CGN, cis-Golgi network; EGF, epidermal growth factor; ER, endoplasmic reticulum; FBS, foetal bovine serum; JNK, Jun kinase; LPA, lysophosphatidic acid; MLC, myosin light chain; mDia 1/2, diaphanous-related; MNK, Menkes disease ATPase; MTOC, microtubule organizing centre; PM, plasma membrane; POR-1, partner of RAC; ROCK, Rho-kinase; ROCK-KIN, constitutively active Rho-kinase; SGC, stack of Golgi cisternae; TfR, transferring receptor; TGN, trans-Golgi network; VSV, Vesicular Stomatitis Virus
CGN, cis-Golgi network; EGF, epidermal growth factor; ER, endoplasmic reticulum; FBS, foetal bovine serum; JNK, Jun kinase; LPA, lysophosphatidic acid; MLC, myosin light chain; mDia 1/2, diaphanous-related; MNK, Menkes disease ATPase; MTOC, microtubule organizing centre; PM, plasma membrane; POR-1, partner of RAC; ROCK, Rho-kinase; ROCK-KIN, constitutively active Rho-kinase; SGC, stack of Golgi cisternae; TfR, transferring receptor; TGN, trans-Golgi network; VSV, Vesicular Stomatitis Virus