RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System.

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Replication deficient, recombinant adenovirus (Ad) vectors do not require target cell replication for transfer and expression of exogenous genes and thus may be useful for in vivo gene therapy in hepatocytes. In vitro, primary cultures of rat hepatocytes infected with a recombinant Ad containing a human alpha 1-antitrypsin cDNA (Ad-alpha 1AT) synthesized and secreted human alpha 1AT for 4 weeks. In rats, in vivo intraportal administration of a recombinant Ad containing the E. coli lacZ gene, was followed by expression of beta-galactosidase in hepatocytes 3 days after infection. Intraportal infusion of Ad-alpha 1AT produced detectable serum levels of human alpha 1AT for 4 weeks. Thus, targeted gene expression has been achieved in the liver, albeit at low levels, suggesting that adenovirus vectors may be a useful means for in vivo gene therapy in liver disorders.

Protein misfolding is monitored by a variety of cellular "quality control" systems. Endoplasmic reticulum (ER) quality control handles misfolded secretory and membrane proteins and is well characterized. However, less is known about the quality control of misfolded cytosolic proteins (CytoQC). To study CytoQC, we have employed a genetic system in Saccharomyces cerevisiae using a transplantable degron, CL1 (1). Attachment of CL1 to the cytosolic protein Ura3p destabilizes Ura3p, targeting it for rapid proteasomal degradation. We have performed a comprehensive analysis of Ura3p-CL1 degradation requirements. As shown previously, we observe that the ER-localized ubiquitin E2 (Ubc6p, Ubc7p, and Cue1p) and E3 (Doa10p) machinery involved in ER-associated degradation (ERAD) are also responsible for the degradation of the cytosolic substrate Ura3p-CL1. Importantly, we find that the cytosol/ER membrane-localized chaperones Ydj1p and Ssa1p, known to be necessary for the ERAD of membrane proteins with misfolded cytosolic domains, are also required for the ubiquitination and degradation of Ura3p-CL1. In addition, we show a role for the Cdc48p-Npl4p-Ufd1p complex in the degradation of Ura3p-CL1. When ubiquitination is blocked, a portion of Ura3p-CL1 is ER membrane-localized. Furthermore, access to the cytosolic face of the ER is required for the degradation of CL1 degroncontaining proteins. The ER is distributed throughout the cytosol, and our data, together with previous studies, suggest that the cytosolic face of the ER membrane serves as a "platform" for the degradation of Ura3p-CL1, which may also be the case for other CytoQC substrates.Mutation, errors in transcription or translation, and cellular stress can cause alterations in amino acids that may prevent proteins from attaining their properly folded, native conformations. Protein "quality control" is an essential process monitoring protein folding, ultimately targeting misfolded proteins for degradation via the ubiquitin-proteasome system. The importance of protein quality control is best exemplified by the numerous human diseases that can result from protein misfolding due to mutational or physiological causes and include cystic fibrosis, Parkinson disease, and ␣ 1 -antitrypsin deficiency (2, 3).Distinct protein quality control systems appear to exist in various cellular compartments, including the nucleus, mitochondria, and endoplasmic reticulum (ER), 2 with the bestcharacterized system being ER quality control (4 -7). Studies of ER quality control and, in particular, ER-associated degradation (ERAD) have revealed discrete chaperone and ubiquitination machinery required for the recognition and ubiquitination of different classes of misfolded secretory or membrane proteins. Much of this work has been greatly aided by the use of "model" ER quality control substrates, such as CPY* or Ste6p*, in the yeast Saccharomyces cerevisiae (8 -12). For example, it has become clear that model membrane proteins with misfolded cytosolic domains (called ERAD-C substrates), such as St...

SUMMARY Cue1p is an integral component of yeast endoplasmic reticulum (ER)-associated degradation (ERAD) ubiquitin ligase (E3) complexes. It tethers the ERAD ubiquitin-conjugating enzyme (E2), Ubc7p, to the ER and prevents its degradation, and also activates Ubc7p via unknown mechanisms. We have now determined the crystal structure of the Ubc7p-binding region (U7BR) of Cue1p with Ubc7p. The U7BR is a unique E2-binding domain that includes three α-helices that interact extensively with the ‘backside’ of Ubc7p. Residues essential for E2 binding are also required for activation of Ubc7p and for ERAD. We establish that the U7BR stimulates both RING-independent and dependent ubiquitin transfer from Ubc7p. Moreover, the U7BR enhances ubiquitin-activating enzyme (E1)-mediated charging of Ubc7p with ubiquitin. This is the first example where an essential component of E3 complexes both binds to E2 and enhances E2 loading with ubiquitin. These findings provide new insights into mechanisms of stimulating ubiquitination.

ER quality control (ERQC) prevents the exit of misfolded secretory and membrane proteins from the ER. A critical aspect of ERQC is a transcriptional response called the unfolded protein response (UPR), which up-regulates genes that enable cells to cope with misfolded, ER-retained proteins. In this study, we compare the transcriptional responses in yeast resulting from the acute expression of misfolded proteins residing in three different cellular compartments (the ER lumen, membrane, and cytosol), and find that each elicits a distinct transcriptional response. The classical UPR response, here-designated UPR-L, is induced by the ER lumenal misfolded protein, CPY*. The UPR-Cyto response is induced by the cytosolic protein, VHL-L158P, and is characterized by a rapid, transient induction of cytosolic chaperones similar to the heat-shock response. In contrast, the misfolded membrane protein with a cystolic lesion, Ste6p*, elicits a unique response designated UPR-M/C, characterized by the modest induction of >20 genes regulated by Rpn4p, an activator of proteasomal genes. Independently, we identified several genes required for yeast viability during UPR-M/C stress, but not UPR-L or UPR-Cyto stress. Among these is RPN4, highlighting the importance of the Rpn4p-dependent response in tolerating UPR-M/C stress. Further analysis suggests the requirement for Rpn4p reflects severe impairment of the proteasome by UPR-M/C stress.

Interleukin-3 (IL-3) is a member of a family of growth factors, each of which supports the proliferation and development of hematopoietic precursors in culture. Although the biologic effects of the different hematopoietic growth factors have been well documented in different culture systems, it has only recently become possible to study the activities of these molecules in vivo. In comparison with the later acting hematopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor, IL-3 elicited a delayed and relatively modest leukocytosis when continuously infused intravenously in primates. The IL-3 infusion, however, greatly potentiated the responsiveness of the animal to subsequent administration of a low dose of GM-CSF. These results suggest that IL-3 expands an early cell population in vivo that subsequently requires the action of a later acting factor such as GM-CSF to complete its development. Optimal stimulation of hematopoiesis may be achieved with combinations of hematopoietic growth factors.

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by ER-associated degradation (ERAD). Although the retrotranslocation of misfolded proteins from the ER has been reconstituted, how a polypeptide is initially selected for ERAD remains poorly defined. To address this question while controlling for the diverse nature of ERAD substrates, we constructed a series of truncations in a single ER-tethered domain. We observed that the truncated proteins exhibited variable degradation rates and discovered a positive correlation between ERAD substrate instability and detergent insolubility, which demonstrates that aggregation-prone species can be selected for ERAD. Further, Hsp104 facilitated degradation of an insoluble species, consistent with the chaperone's disaggregase activity. We also show that retrotranslocation of the ubiquitinated substrate from the ER was inhibited in the absence of Hsp104. Therefore, chaperone-mediated selection frees the ER membrane of potentially toxic, aggregation-prone species.