Autophagy is a major catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles. This pathway is activated under environmental stress conditions, during development and in various pathological situations. In this study, we describe the role of reactive oxygen species (ROS) as signaling molecules in starvation-induced autophagy. We show that starvation stimulates formation of ROS, specifically H 2 O 2 . These oxidative conditions are essential for autophagy, as treatment with antioxidative agents abolished the formation of autophagosomes and the consequent degradation of proteins. Furthermore, we identify the cysteine protease HsAtg4 as a direct target for oxidation by H 2 O 2 , and specify a cysteine residue located near the HsAtg4 catalytic site as a critical for this regulation. Expression of this regulatory mutant prevented the formation of autophagosomes in cells, thus providing a molecular mechanism for redox regulation of the autophagic process.

The insulin receptor (IR)1 is a heterotetrameric transmembrane glycoprotein composed of two extracellular ␣ subunits and two transmembrane ␤ subunits linked by disulfide bonds. The ␣ subunits contain the insulin-binding domain, while the transmembrane ␤ subunits function as Tyr-specific kinases (insulin receptor kinases). Insulin signaling utilizes the Tyr kinase activity of the receptor to phosphorylate docking proteins on multiple Tyr residues and further propagate insulin action (1). The major substrates of insulin receptor kinase are Shc (2) and the IRS proteins, IRS-1 (3), IRS-2 (4), IRS-3 (5), and IRS-4 (6). IRS proteins contain a conserved pleckstrin homology domain (7, 8) located at the amino terminus, adjacent to a phosphotyrosine binding (PTB) domain. The PTB domain is present in a number of signaling molecules (9) and shares 75% sequence identity between IRS-1 and IRS-2 (10). This domain interacts with the NPXY motif of the juxtamembrane (JM) region of IR and promotes IR/IRS-1 interactions (11, 12). The C-terminal region of IRS proteins is poorly conserved. It contains multiple Tyr phosphorylation motifs that serve as docking sites for SH2 domain-containing proteins like the p85␣ regulatory subunit of PI3K, Grb2, Nck, Crk, Fyn, SHP-2, and others, which mediate the metabolic and growth-promoting functions of insulin (1, 13).The signaling pathways regulated by IRS proteins control glucose uptake and lipogenesis, protein synthesis, and cell survival (1, 13). The relative roles of the different IRS proteins in mediating insulin action are still unclear; however, studies of gene disruption revealed that IRS-2 compensates for the absence of IRS-1 in hepatocytes of IRS-1 null mice, while IRS-3 provides the major alternative pathway to PI3K activation in skeletal muscle and adipocytes of these animals (14 -17). In contrast, IRS-2 null mice develop both insulin resistance and beta cell failure, which leads to their death (18). These data implicate different IRS proteins as mediators of insulin action in different tissues.IRS proteins contain over 30 potential Ser/Thr phosphorylation sites for kinases like protein kinase A, PKC, and mitogenactivated protein kinase (3,4,19). In previous studies, we have demonstrated that Ser/Thr phosphorylation of IRS-1 and IRS-2 significantly reduces their ability to interact with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate insulin signaling, thus providing a possible molecular mechanism for the induction of an insulinresistant state (20,21). Ser/Thr phosphorylation of IRS pro-

The nuclear envelope of higher eukaryotic cells reforms at the exit from mitosis, in concert with the assembly of nuclear pore complexes (NPCs). The first step in postmitotic NPC assembly involves the "seeding" of chromatin with ELYS and the Nup107-160 complex. Subsequent steps in the assembly process are poorly understood and different mechanistic models have been proposed to explain the formation of the full supramolecular structure. Here, we show that the initial step of chromatin seeding is negatively regulated by importin beta. Direct imaging of the chromatin attachment sites reveals single sites situated predominantly on the highest substructures of chromatin surface and lacking any sign of annular structures or oligomerized pre-NPCs. Surprisingly, the inhibition by importin beta is only partially reversed by RanGTP. Importin beta forms a high-molecular-weight complex with both ELYS and the Nup107-160 complex in cytosol. We suggest that initiation sites for NPC assembly contain single copies of chromatin-bound ELYS/Nup107-160 and that the lateral oligomerization of these subunits depends on the recruitment of membrane components. We predict that additional regulators, besides importin beta and Ran, may be involved in coordinating the initial seeding of chromatin with subsequent steps in the NPC assembly pathway.

Docking of a vesicle at the appropriate target membrane involves an interaction between integral membrane proteins located on the vesicle (v-SNAREs) and those located on the target membrane (t-SNAREs). GATE-16 (Golgi-associated ATPase enhancer of 16 kDa) was shown to modulate the activity of SNAREs in the Golgi apparatus and is therefore an essential component of intra-Golgi transport and post-mitotic Golgi reassembly. GATE-16 contains a ubiquitin fold subdomain, which is terminated at the carboxyl end by an additional amino acid after a conserved glycine residue. In the present study we tested whether the COOH terminus of GATE-16 undergoes post-translational cleavage by a protease which exposes the glycine 116 residue. We describe the isolation and characterization of HsApg4A as a human protease of GATE-16. We show that GATE-16 undergoes COOH-terminal cleavage both in vivo and in vitro, only when the conserved glycine 116 is present. We then utilize an in vitro assay to show that pure HsApg4A is sufficient to cleave GATE-16. The characterization of this protease may give new insights into the mechanism of action of GATE-16 and its other family members.

Nuclear targeting of intact proteasome particles was tested in the Xenopus egg extract system. Both the 26S proteasome holoenzyme and the 20S core particle were targeted to the nuclear envelope but could not enter the nucleus. A novel proteolytically active 20S+ particle was actively imported into the nucleoplasm in a Ran-independent fashion.