Small ubiquitin-related modifier (SUMO) processing and deconjugation are mediated by sentrin-specific proteases/ubiquitin-like proteases (SENP/Ulps). We show that SUMO-specific protease 1 (SUSP1), a mammalian SENP/Ulp, localizes within the nucleoplasm. SUSP1 depletion within cell lines expressing enhanced green fluorescent protein (EGFP) fusions to individual SUMO paralogues caused redistribution of EGFP-SUMO2 and -SUMO3, particularly into promyelocytic leukemia (PML) bodies. Further analysis suggested that this change resulted primarily from a deficit of SUMO2/3-deconjugation activity. Under these circumstances, PML bodies became enlarged and increased in number. We did not observe a comparable redistribution of EGFP-SUMO1. We have investigated the specificity of SUSP1 using vinyl sulfone inhibitors and model substrates. We found that SUSP1 has a strong paralogue bias toward SUMO2/3 and that it acts preferentially on substrates containing three or more SUMO2/3 moieties. Together, our findings argue that SUSP1 may play a specialized role in dismantling highly conjugated SUMO2 and -3 species that is critical for PML body maintenance.

Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain-containing protein 1 (DFCP1)-positive omegasome and the IM accumulated in Atg3-deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron microscopy and immunoelectron microscopy revealed that green fluorescent protein (GFP)-tagged DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims. Through detailed morphological analyses, including optimization of a fixation method and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/or ER. The formation of these thin tubular clusters was observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1 but not in FIP200-deficient cells, suggesting that they were relevant to the earlier events in autophagosome formation. Taken together, our findings indicate that these tubular profiles represent a part of the omegasome that links the ER with the IM.

In a previous study, we demonstrated that the forkhead associated (FHA) domain of pKi-67 interacts with the novel kinesin-like protein, Hklp2 (Sueishi, M., Takagi, M., and Yoneda, Y. (2000) J. Biol. Chem. 275, 28888 -28892). In this study, we report on the identification of a putative RNA-binding protein of 293 residues as another binding partner of the FHA domain of pKi-67 (referred to as NIFK for nucleolar protein interacting with the FHA domain of pKi-67). Human NIFK (hNIFK) interacted with the FHA domain of pKi-67 (Ki-FHA) efficiently in vitro when hNIFK was derived from mitotically arrested cells. In addition, a moiety of hNIFK was co-localized with pKi-67 at the peripheral region of mitotic chromosomes. The hNIFK domain that interacts with Ki-FHA was mapped in the yeast two-hybrid system to a portion encompassed by residues 226 -269. In a binding assay utilizing Xenopus egg extracts, it was found that the mitosis-specific environment and two threonine residues within this portion of hNIFK (Thr-234 and Thr-238) were crucial for the efficient interaction of hNIFK and Ki-FHA, suggesting that hNIFK interacts with Ki-FHA in a mitosis-specific and phosphorylation-dependent manner. These findings provide a new clue to our understanding of the cellular function of pKi-67.The Ki-67 antigen (pKi-67), originally identified as the antigen for a monoclonal antibody raised against the nuclear extract from a Hodgkin's lymphoma-derived cell line, was characterized as a class of proteins that localize around mitotic chromosomes (1). As a result, it is assumed that pKi-67 is involved in mitotic chromosome organization. pKi-67 is a convenient cell proliferation marker, since its expression is restricted to growing cells (2). Although the recent identification and characterization of a marsupial counterpart of pKi-67, which is referred to as chmadrin, suggests that pKi-67 plays some type of role in the organization of higher order chromatin structure (3), the actual role of pKi-67 in the cell cycle progression remains unclear.The N-terminal portion of pKi-67 is well conserved between human pKi-67 and chmadrin (62% identical) and contains a forkhead associated (FHA) 1 domain. It was originally reported that the FHA domain constituted a region that has been conserved in a subset of forkhead-type transcription factors (4). The sequence profile has been reported for a variety of proteins with diverse functions (transcription, DNA repair, cell cycle progression, etc.). In several instances, the FHA domain preferentially recognizes partner proteins when they are present in the phosphorylated form (5-8). Moreover, the strong specificity of the FHA domain for phosphopeptides has been clearly demonstrated by binding assays with synthetic phosphopeptides (9). Therefore, it is currently thought that the FHA domain is a general phosphopeptide recognition motif that is involved in certain phosphopeptide-mediated signal transduction pathways (10). A search for the interaction partner(s) of the FHA domain of pKi-67, which could exist in the...

Background: The LR domain of marsupial chmadrin is defined by its C‐terminal amino acid sequence, which contains several pairs of leucine (L) and arginine (R) residues. The LR domain of chmadrin causes a significant compaction of chromatin over the entire length of chromosomes when it is overproduced. The possible human homologue of chmadrin, Ki‐67 antigen (pKi‐67), also has a stretch of LR pairs, but with no obvious overall similarity, at its C‐terminus. Results: The LR domain of human pKi‐67 also induced chromatin compaction, both in human and marsupial cells. A yeast two‐hybrid assay and an in vitro binding assay demonstrated that the human LR domain binds to heterochromatin protein 1 (HP1), a well‐characterized molecule as a mediator of heterochromatin formation. In fixed cells stained with specific antibodies, the pKi‐67 was found to be co‐localized partially with HP1 at foci on chromosomes in an early G1 phase. Time‐lapse observation in living cells co‐expressing the fluorescently tagged proteins showed that the LR domain formed foci on chromosomes over a limited period of the cell cycle from the telophase to early G1 phase and that HP1 subsequently accumulated at these foci of the LR domain. Conclusions: Marsupial chmadrin and human pKi‐67 induce chromatin compaction across species, possibly via the interaction of its LR domain with HP1.