Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation.

Eukaryotic cells rely on a surveillance mechanism known as the spindle checkpoint to ensure accurate chromosome segregation. The spindle checkpoint prevents sister chromatids from separating until all kinetochores achieve bipolar attachments to the mitotic spindle. Checkpoint proteins tightly inhibit the anaphase-promoting complex (APC), a ubiquitin ligase required for chromosome segregation and progression to anaphase. Unattached kinetochores promote the binding of checkpoint proteins Mad2 and BubR1 to the APC-activator Cdc20, rendering it unable to activate APC. Once all kinetochores are properly attached, however, cells inactivate the checkpoint within minutes, allowing for the rapid and synchronous segregation of chromosomes. How cells switch from strong APC inhibition before kinetochore attachment to rapid APC activation once attachment is complete remains a mystery. Here we show that checkpoint inactivation is an energy-consuming process involving APC-dependent multi-ubiquitination. Multi-ubiquitination by APC leads to the dissociation of Mad2 and BubR1 from Cdc20, a process that is reversed by a Cdc20-directed de-ubiquitinating enzyme. The mutual regulation between checkpoint proteins and APC leaves the cell poised for rapid checkpoint inactivation and ensures that chromosome segregation promptly follows the completion of kinetochore attachment. In addition, our results suggest a mechanistic basis for how cancer cells can have a compromised spindle checkpoint without corresponding mutations in checkpoint genes.

Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone.

The OLE pathway of yeast regulates the level of the ER-bound enzyme Delta9-fatty acid desaturase OLE1, thereby controlling membrane fluidity. A central component of this regulon is the transcription factor SPT23, a homolog of mammalian NF-kappaB. SPT23 is synthesized as an inactive, ER membrane-anchored precursor that is activated by regulated ubiquitin/proteasome-dependent processing (RUP). We now show that SPT23 dimerizes prior to processing and that the processed molecule, p90, retains its ubiquitin modification and initially remains tethered to its unprocessed, membrane-bound SPT23 partner. Subsequently, p90 is liberated from its partner for nuclear targeting by the activity of the chaperone-like CDC48(UFD1/NPL4) complex. Remarkably, this enzyme binds preferentially ubiquitinated substrates, suggesting that CDC48(UFD1/NPL4) is qualified to selectively remove ubiquitin conjugates from protein complexes.

Membrane-bound transcription factors: regulated release by RIP or RUP.

Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membrane-bound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane.

Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.

Processing of integral membrane proteins in order to liberate active proteins is of exquisite cellular importance. Examples are the processing events that govern sterol regulation, Notch signaling, the unfolded protein response, and APP fragmentation linked to Alzheimer's disease. In these cases, the proteins are thought to be processed by regulated intramembrane proteolysis, involving site-specific, membrane-localized proteases. Here we show that two homologous yeast transcription factors SPT23 and MGA2 are made as dormant ER/nuclear membrane-localized precursors and become activated by a completely different mechanism that involves ubiquitin/proteasome-dependent processing. SPT23 and MGA2 are relatives of mammalian NF-kappaB and control unsaturated fatty acid levels. Intriguingly, proteasome-dependent processing of SPT23 is regulated by fatty acid pools, suggesting that the precursor itself or interacting partners are sensors of membrane composition or fluidity.

Recognition of protein substrates by the prolyl isomerase trigger factor is independent of proline residues.

The trigger factor is associated with bacterial ribosomes and catalyzes proline-limited protein folding reactions. Its folding activity is very high and conserved in evolution, as shown for the homologous enzymes from Escherichia coli and Mycoplasma genitalium. The folding protein substrate (a variant of ribonuclease T1) binds with high affinity to the trigger factors, and permanently unfolded proteins are strong, competitive inhibitors. We used this inhibition to characterize the substrate binding sites of the trigger factors. Unfolded alpha-lactalbumin binds very tightly and inhibits the trigger factor from M. genitalium with a KI value of 50 nM. The binding of inhibitory proteins is independent of proline residues, as shown for unfolded tendamistat, which binds to the trigger factor with equal affinity in the presence and in the absence of its three proline residues. The good inhibition by a non-folding variant of ribonuclease T1 that lacks Pro39 showed that this proline, at which the catalysis of folding occurs, is dispensable for substrate binding. The trigger factors cannot catalyze prolyl isomerization when proteins are partially folded already. They preferentially recognize unstructured protein chains, which bind with high affinity to a site distinct from the catalytic prolyl isomerase center in the FKBP domain.