Human SGT interacts with Bag-6/Bat-3/Scythe and cells with reduced levels of either protein display persistence of few misaligned chromosomes and mitotic arrest

Abstract

The human small glutamine-rich TPR-containing protein (hSGT) is essential for cell division since RNA-interference-mediated strong reduction of hSGT protein levels causes mitotic arrest (M. Winnefeld, J. Rommelaere, and C. Cziepluch, The human small glutamine-rich TPR-containing protein is required for progress through cell division, Exp. Cell Res. 293 (2004), 43–57). Analysis of HeLa cells expressing a histone 2A-YFP fusion protein revealed the continuous presence of few mislocalized chromosomes close to the spindle poles as possible cause for hSGT depletion-dependent prometaphase arrest. Cells unable to rescue these mislocalized chromosomes into the metaphase plate died at this stage through apoptosis. In order to address hSGT function at the molecular level, mass spectrometry analysis of proteins which co-immunoprecipitated with Flag-tagged hSGT was performed. Thereby, Hsp70 and Bag-6/Bat-3/Scythe were identified as novel hSGT interaction partners while interaction with Hsc70 was confirmed. Results obtained with truncated versions of the hSGT protein revealed that Bag-6/Bat-3/Scythe and Hsp70 or Hsc70 were independently able to form complexes with hSGT. Interaction of hSGT with Hsc70, Hsp70 or Bag-6/Bat-3/Scythe was demonstrated in prometaphase, thereby suggesting a possible role for complexes containing hSGT and distinct (co)-chaperones during mitosis. Finally, cells from populations with reduced levels of Bag-6/Bat-3/Scythe also displayed persistence of mislocalized chromosomes and mitotic arrest, which strongly indicated that hSGT-Bag-6/Bat-3/Scythe complexes could be directly or indirectly required for complete chromosome congression.

Introduction

Mitosis and cytokinesis are central processes in cell division through which the cell coordinates the separation of sister chromatids and their distribution into the emerging two daughter cells, thus ensuring that both daughter cells will contain a full set of genetic information. The importance of these processes is further highlighted by the fact that genetic defects causing disturbances in these processes contribute to the development of cancer and that on the other hand molecules involved in mitosis constitute effective classical or promising new targets for cancer therapy [2], [3], [4], [5], [6]. Therefore, great efforts are being made to understand mitosis and cytokinesis at the molecular level. Many of the involved cellular factors are known, although their detailed molecular function is not fully understood in all cases. In addition, essential factors are still being identified.

We have recently discovered that the human small glutamine-rich TPR-containing protein (hSGT) is essential for cell division [1]. SGT is a housekeeping protein [1], [7] with a tripartite structure. The N-terminal domain confers homo-oligomerization [7], [8], a centrally located TPR domain in conjunction with neighboring C-terminal residues is responsible for interaction with Hsc70 [9], [10], [11], [12], and the C-terminal part shows an accumulation of glutamine residues [13]. So far, most studies concerning SGT function have been conducted in neuronal cells where a stable complex composed of SGT, Hsc70 and the co-chaperon cystein string protein (Csp) which is a neuron-specific J-domain protein was identified. In vitro studies show that interaction with SGT and Csp increases Hsp70 ATPase and luciferase refolding activity [12]. More recent results suggest that the Csp/Hsp70/SGT complex may actually also function as a GDP/GTP exchange factor for Gαs subunits of GTP binding proteins and that Csp nucleotide exchange activity is regulated by Hsc70 and SGT [14].

The ubiquitous presence of SGT, however, implied that this protein also has additional functions apart from the ones in neurons. As mentioned above, we have addressed this issue in a previous study using an siRNA-based approach. In all human cell lines tested, hSGT depletion specifically caused reduced proliferation. In the transformed human new born kidney epithelial cell line (NBE), this was due to hSGT depletion-specific arrests in mitosis, defects in cytokinesis and the occurrence of M-phase-specific cell death. Moreover, hSGT localization also supports a role for this protein in cell division. In prometa- and metaphase, hSGT is detected at the spindle poles. During anaphase, hSGT relocates to the central spindle where pole microtubules overlap. Throughout telophase and into early G1, hSGT accumulates in the midbody. Finally, hSGT displays a mitosis-specific phosphorylation pattern pointing to a possible regulation of this protein during mitosis [1].

Aim of the present study was to determine the trigger for mitotic arrest in hSGT-depleted cells. Time-lapse video microscopy of HeLa cells expressing a H2A-YFP fusion protein revealed that hSGT-depleted cells stayed arrested in prometaphase with most chromosomes aligned in the metaphase plate. The continuous presence of few mislocalized chromosomes close to the spindle poles was identified as at least one main reason for mitotic arrest. To initiate hSGT functional analysis at the molecular level, we determined binding partners for hSGT. MALDI-TOF mass spectrometry analysis of hSGT co-immunoprecipitating proteins identified Bag-6/Bat-3/Scythe, Hsc70 and Hsp70 as endogenous interaction partners. Stable complexes of these proteins were formed during interphase but also in prometaphase and could therefore directly or indirectly contribute to correct chromosome alignment during this stage of mitosis. This assumption was supported by the finding that depletion of Bag-6/Bat-3/Scythe also led to the appearance of mislocalized chromosomes, mitotic arrest and subsequent cell death.