AP-1 clathrin adaptor and CG8538/Aftiphilin are involved in Notch signaling during eye development in Drosophila melanogaster.

Clathrin adaptor protein complex-1 (AP-1) and its accessory proteins play a role in the sorting of integral membrane proteins at the trans-Golgi network and endosomes. Their physiological functions in complex organisms, however, are not fully understood. In this study, we found that CG8538p, an uncharacterized Drosophila protein, shares significant structural and functional characteristics with Aftiphilin, a mammalian AP-1 accessory protein. The Drosophila Aftiphilin was shown to interact directly with the ear domain of γ-adaptin of Drosophila AP-1, but not with the GAE domain of Drosophila GGA. In S2 cells, Drosophila Aftiphilin and AP-1 formed a complex and colocalized at the Golgi compartment. Moreover, tissue-specific depletion of AP-1 or Aftiphilin in the developing eyes resulted in a disordered alignment of photoreceptor neurons in larval stage and roughened eyes with aberrant ommatidia in adult flies. Furthermore, AP-1-depleted photoreceptor neurons showed an intracellular accumulation of a Notch regulator, Scabrous, and downregulation of Notch by promoting its degradation in the lysosomes. These results suggest that AP-1 and Aftiphilin are cooperatively involved in the intracellular trafficking of Notch during eye development in Drosophila.

SUSP1 antagonizes formation of highly SUMO2/3-conjugated species.

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.

A cluster of thin tubular structures mediates transformation of the endoplasmic reticulum to autophagic isolation membrane.

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.

Interaction of the chromatin compaction-inducing domain (LR domain) of Ki-67 antigen with HP1 proteins.

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.