Summary In E1-E2-E3 ubiquitin (Ub) conjugation cascades, the E2 first forms a transient E2~Ub covalent complex, and then interacts with an E3 for Ub transfer. For cascades involving E3s in the HECT class, Ub is transferred from an associated E2 to the acceptor Cys in the HECT domain C-lobe. To gain insights into this process, we determined the crystal structure of a complex between the HECT domain of NEDD4L and the E2 UbcH5B bearing a covalently-linked Ub at its active site (UbcH5B~Ub). Noncovalent interactions between UbcH5B and the HECT N-lobe and between Ub and the HECT domain C-lobe lead to an overall compact structure, with the Ub C-terminus sandwiched between UbcH5B and HECT domain active sites. The structure suggests a model for E2-to-HECT Ub transfer, in which interactions between a donor Ub and an acceptor domain constrain upstream and downstream enzymes for conjugation.

While ESCRT-0 is ubiquitinated by the Rsp5 E3 ligase, loss of Rsp5 does not disrupt monoubiquitin-dependent sorting into multivesicular bodies.

The efficient formation of a variety of transport vesicles is influenced by the presence of cargo, suggesting that cargo itself might have a defining role in vesicle biogenesis. However, definitive in vivo experiments supporting this concept are lacking, as it is difficult to eliminate endogenous cargo. The Endosomal Sorting Complexes Required for Transport (ESCRT) apparatus sorts ubiquitinated membrane proteins into endosomal intralumenal vesicles (ILVs) that accumulate within multivesicular bodies. Here we show that cargo ubiquitination is required for effective recruitment of the ESCRT machinery onto endosomal membranes and for the subsequent formation of ILVs.

The process in which ubiquitin (Ub) conjugation is required for trafficking of integral membrane proteins into multivesicular bodies (MVBs) and eventual degradation in the lumen of lysosomes/vacuoles is well-defined. However, Ub-independent pathways into MVBs are less understood. To better understand this process, we have further characterized the membrane protein Sna3, the prototypical Ub-independent cargo protein sorted through the MVB pathway in yeast. We show that Sna3 trafficking to the vacuole is critically dependent on Rsp5 ligase activity and ubiquitination. We find Sna3 undergoes Ub-dependent MVB sorting either by becoming ubiquitinated itself or associating with other ubiquitinated membrane protein substrates. In addition, our functional studies support a role for Sna3 as an adaptor protein that recruits Rsp5 to cargo such as the methionine transporter Mup1, resulting in efficient Mup1 delivery to the vacuole.

p21-activated protein kinases (PAKs) are a family of serine/threonine protein kinases that are activated by binding of the p21 G proteins Cdc42 or Rac. The ubiquitous PAK-2 (␥-PAK) is unique among the PAK isoforms because it is also activated through proteolytic cleavage by caspases or caspase-like proteases. In response to stress stimulants such as tumor necrosis factor ␣ or growth factor withdrawal, PAK-2 is activated as a fulllength enzyme and as a proteolytic PAK-2p34 fragment. Activation of full-length PAK-2 stimulates cell survival, whereas proteolytic activation of PAK-2p34 is involved in programmed cell death. Here we provide evidence that the proapoptotic effect of PAK-2p34 is regulated by subcellular targeting and degradation by the proteasome. Full-length PAK-2 is localized in the cytoplasm, whereas the proteolytic PAK-2p34 fragment translocates to the nucleus. Subcellular localization of PAK-2 is regulated by nuclear localization and nuclear export signal motifs. A nuclear export signal motif within the regulatory domain prevents nuclear localization of fulllength PAK-2. Proteolytic activation removes most of the regulatory domain and disrupts the nuclear export signal. The activated PAK-2p34 fragment contains a nuclear localization signal and translocates to the nucleus. However, levels of activated PAK-2p34 are tightly regulated through ubiquitination and degradation by the proteasome. Inhibition of degradation by blocking polyubiquitination results in significantly increased levels of PAK-2p34 and as a consequence, in stimulation of programmed cell death. Therefore, nuclear targeting and inhibition of degradation appear to be critical for stimulation of the cell death response by PAK-2p34.