Proteins of the BTB-kelch family are known to be involved in multiple biological processes such as migration, cytoskeleton arrangement, regulation of cell morphology, protein ubiquitination and gene expression
. Therefore a prediction about the function of a new member of this family is difficult. Here we describe a novel BTB-kelch protein, KBTBD8 that is highly conserved from mouse to human. We report that 1) Kbtbd8 is ubiquitiously expressed; 2) KBTBD8 is a novel protein of the BTB-kelch family that is located in the Golgi apparatus; 3) KBTBD8 is not a Golgi-resident protein and 4) KBTBD8 is the first BTB-kelch protein that localizes on the spindle apparatus upon mitosis in proliferating cells.
There are many BTB-subfamilies known that are distributed through species but the quantity of families and their members varies between species (reviewed in
). Whereas for Saccharomyces cerevisiae there are only four subfamilies with six proteins known, Drosophila melanogaster already harbours eight subfamilies with 85 proteins. Mus musculus and Homo sapiens have the same eight subfamilies with 194 and 183 total proteins, respectively. The subfamilies are known as BTB only, T1-Kv, ElonginC, Skp1, MATH-BTB, other architectures, BTB-ZF and BBK whereas the last two resemble the largest groups with more than 40 proteins in mouse and human.
BTB-ZF (BTB-Zinc finger) proteins are also known as POK (POZ and Krüppel zinc finger) proteins
. As expected for zinc finger proteins, many members of this family were characterized as transcription factors. The BTB- domain in these proteins is responsible for dimerisation or even for heteromeric BTB-BTB associations whereas the ZFs are responsible for DNA binding.
The BBK (or BTB-BACK-kelch) family resembles the second large group within the BTB proteins. The architecture of these proteins, the BTB/POZ domain together with the BACK domain at the N-terminal part of the proteins and the kelch-repeats at the C-terminal part of the proteins, allows on the one hand the dimerization via the BTB domain and on the other hand the association to cytoskeleton via their kelch-repeat domain, which is known for many BBK proteins
. As Kbtbd8 belongs to this family and we could observe faint cytoskeleton staining in rare cases, we co-stained with tubulin (Figure
4) in Nocodazole treated and untreated cells but could not confirm this assumption. Therefore, Kbtbd8 seems not to be associated to the cytoskeleton.
In non-dividing cells, the Golgi apparatus is located at the periphery of the nucleus, close to the ER. During cell division the Golgi needs to be segregated and distributed to the two daughter cells. With the onset of mitosis, the Golgi complex become fragmented and disperses throughout the cell. The Golgi matrix associated proteins like GM130 mark this dispersion and can be found as punctuated structures throughout the cytoplasm. Treatment of cells with Brefeldin A (BFA) has the same effect: it blocks the protein transport from the ER to the Golgi
 and therefore causes a collapse of the Golgi stacks resulting in punctuated structures. Furthermore it was shown that treatment with BFA had no effect on partitioning of Golgi matrix proteins during mitosis. GM130 positive matrix fragment become associated with the forming spindle poles in pro- and metaphase stages. In anaphase cells, these fragments were found to move with the separating sister chromatids and end up on both sides of each new-forming nucleus
. Furthermore, it was shown that the spindle plays a direct role in the inheritance of the Golgi
To our knowledge there is only one other BTB-kelch protein known, that shows the golgi compartment as its subcellular localization: “leucin zipper-like transcriptional regulator 1” (LZTR-1). Nacak et al.
 characterized LZTR-1 and could show a Golgi matrix associated behaviour of LZTR-1 upon BFA treatment and therefore conclude that LZTR-1 is, as GM130, a golgi matrix protein. They further analyzed the domains concerning their function and could show that the BTB domain is responsible for the localization in the golgi apparatus. Since KBTBD8 shows a different behaviour upon BFA treatment than GM130, we conclude that KBTBD8 is not a matrix associated Golgi protein but trapped in the lumen. These results fit to the observation that KBTBD8 does not show a 100% co-localisation with GM130, 58 K and Golgin97. Upon dissociation of the Golgi complex during the entry into mitosis, KBTBD8 seems to be released and translocated to the spindle apparatus as we could show by co-staining with α-tubulin on mitotic cells.
Although there is no protein from the BTB-kelch family known that shows a connection between the Golgi complex and the microtubule network, other proteins have been shown to be associated with both, the cytoskeleton – either actin filaments or microtubules - and the Golgi. These proteins include members of the Hook families
, cytoplasmic linker proteins (CLIPs)
, and plakins
. The Microtubule actin crosslinking factor 1b (MACF1b) was shown to be a linker protein between the actin filaments and the microtubules. However, MACF1b is - despite its localization to the cytoskeleton – mainly located in the Golgi and it could be shown that the altered expression of MACF1b mediated by RNA interference resulted in the dispersal of the Golgi complex. A similar dislocation of Golgi structures could be also seen by KBTBD8 downregulation. These results lead us to suggest that KBTBD8 plays an important role in the maintenance of the Golgi structure. Whether altered KBTBD8 expression will also affect the mitotic spindle, needs to be elucidated in the future.