Details of destruction, one molecule at a time

单细胞计数

homeostasis and cell physiology.

Precisely how SRP and NAC interact at the

ribosome remains unclear. Gamerdinger et

al. propose that SRP and NAC compete for

overlapping binding sites on the ribosomal

protein uL23 ( 4), and that NAC binds trans-

lating ribosomes, unless an emerging signal

sequence provides a selective binding ad-

vantage to SRP. Other in vitro data suggest

that there is an alternative NAC ribosome

binding site near eL31 ( 5, 12), and that both

NAC and SRP concomitantly bind the ribo-

some ( 12). SRP could quickly scan translat-

ing ribosomes irrespective of NAC presence,

until an emerging signal sequence triggers

strong SRP binding and NAC release. How

other ribosome-bound chaperones and en-

zymes involved in the folding and process-

ing of nascent chains affect the selection of

NAC versus SRP also remains unclear. NAC

apparently directly influences cotranslational

import of proteins into mitochondria in yeast

( 14), possibly explaining induction of the mi-

tochondrial stress response upon NAC deple-

tion seen in the C. elegans study.

The in vivo work by Gamerdinger et al.

establishes and further defines a central

process in protein biogenesis for metazoan

cells, and corroborates much of the earlier in

vitro work done by Wiedmann. Systematic

approaches such as proteome-wide inter-

action profiling of nascent chains are now

needed to elucidate the dynamics and in-

terplay of SRP, NAC, and other ribosome-

associated factors at the ribosome. Finally,

the Deuerling-Wiedmann model (see the

figure) of antagonistic “sort and countersort”

reflects a recurring principle of check and

countercheck common to a number of bio-

logical mechanisms. Such systems provide a

calibrated equilibrium between two oppos-

ing functions that enhances accuracy and ef-

ficiency in decision-making processes within

living cells. ■

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10.1126/science.aab1335

SCIENCE http://wendang.chazidian.comdeterminants for substrate engagement by the proteasome, and delineated the mecha-ssential cellular processes, such as nism occurring within the proteasome that cell division, rely on the coordinated couples the initiation of protein degrada-destruction of proteins. The predomi-tion with the removal of ubiquitin from the nant means of accomplishing this in-substrate.volves a large cellular machine, the The APC/C has a difficult task. It needs proteasome ( 1). Proteasomal degrada-to precisely and quickly identify proteins tion ensues when proteins are modified with for disposal for the cell cycle to proceed. ubiquitin, a small protein, that has many For this purpose, the APC/C utilizes short, different roles ( 2). This tagging involves a low-complexity recognition sequences in its carrier protein (an E2 ubiquitin-conjugating substrates, which it binds to with the help enzyme) and a substrate-determining pro-of coactivators ( 3). Because these sequences tein (an E3 ligase). For example, during the are present in roughly one-third of the cell’s cell division cycle, a large multiprotein E3 entire protein repertoire (“proteome”), it is ligase, the anaphase-promoting complex/cy-unclear how the APC/C distinguishes po-closome (APC/C), utilizes two E2 enzymes, tential substrates. Nonetheless, once APC/C UBE2C and UBE2S, to target proteins for selects a substrate, it is ubiquitinated and destruction ( 3). On pages 199 and 200 of degraded within minutes.this issue, two Research Articles by Lu et Lu et al. (4) find that the first encounter of al. focus on these reactions and illuminate, the APC/C with a substrate leads to efficient at the single-molecule level, the process of mono-, di-, and triubiquitin modification on ubiquitination by APC/C ( 4), as well as the multiple sites (lysine residues), driven by recognition and subsequent destruction of APC/C substrates by proteasomes ( 5). Both studies substantially enrich our knowledge of ubiquitination and degradation, reveal new properties of APC/C and the protea-some, and challenge established concepts about the ubiquitin-proteasome system.In one study, Lu et al. ( 4) immobilized fluorescently labeled APC/C substrates on a glass slide and then exposed the slide to APC/C. Interaction between an APC/C and UBE2C. This was observed both with puri-a substrate, and the subsequent attachment fied components, but remarkably also with of fluorescent ubiquitin to the substrate, endogenous APC/C activity from cell lysates. were analyzed using total internal reflec-Intriguingly, the authors found that after ini-tion fluorescence (TIRF) microscopy. In the tial ubiquitination, affinity of the substrate other study, the authors analyzed interac-for the APC/C is increased. This indicates tions between immobilized fluorescently la-that there may be unknown ubiquitin recep-beled proteasomes and a range of substrates tors on the APC/C, and recent insight into containing chains of fluorescent ubiquitin the APC/C from structural biology ( 6) should of defined length and composition. In both facilitate their identification. Moreover, studies, the fluorescently labeled ubiquitin based on the observed interaction with ubiq-allowed reporting on the number of ubiqui-uitinated substrates, the authors propose a tin moieties attached to the substrates. The feedforward-like mechanism called “pro-approach has enabled a kinetic description cessive affinity amplification” (see the fig-of the ubiquitin transfer reaction, revealed ure), which ensures that substrates marked for destruction are kept in a ubiquitinated state, while ubiquitinatable decoy un-sub-Medical Research Council Laboratory of Molecular Biology, strates can be selected against by the APC/C. Francis Crick Avenue, Cambridge CB2 0QH, UK. E-mail: dk@mrc-lmb.cam.ac.ukDespite multiple encounters and higher By David Komander “Both studies … challenge established concepts about the ubiquitin-proteasome system.”10 APRIL 2015 ? VOL 348 ISSUE 6231 183

Published by AAAS

单细胞计数

INSIGHTS | PERSPECTIVES

Efcient degradation

Protein

Multi mono-Ub

Highly

processive

initiation

APC/CProteasome

Multi di-Ub

Processive afnity (best substrate)

amplifcation

Mono-UbDeubiquitination prior

Short chainsto degradation

Single long Ub chain

Optimizing demise. APC/C maintains an ubiquitination level suitable for protein destruction (left). Those tagged with multiple short chains are superior proteasome substrates (right).affinity for APC/C, preubiquitinated sub-the cooperativity presumably originates requirement, in needing enough ubiquitin strates are not efficiently modified beyond from engaging separate receptors for ubiq-to establish sufficient residence times on its the first set of ubiquitins; chain elongation uitin, the subsequent affinity increases are ubiquitin receptors, as well as a qualitative by UBE2S is comparatively inefficient. This likely due to avidity effects.requirement in processing only substrates suggests that reassociation with the APC/C Why then are multi-monoubiquitinated modified with ubiquitin chains, which are may serve to simply “top up” ubiquitination proteins not degraded by the proteasome? essential to initiate degradation. It makes to keep substrates primed for proteasomal Indeed, the single-molecule dwell-times of perfect sense that the APC/C focuses on en-degradation. However, the latter result is multi-mono- and polyubiquitinated proteins suring that the minimal requirements are somewhat inconsistent with findings that at the proteasome are similar. Lu et al. (5) find met for its multiple substrates, as such effi-an APC/C substrate binding event leads that a ubiquitin chain, irrespective of length, ciency is likely important to coordinate the to processive ubiquitin amplification by has to be present to activate proteasomal fundamental processes of cell division.

UBE2S ( 7, 8).degradation. The first step in degradation is The idea that proteasomal degradation The observed APC/C activity is consistent the initiation of translocating a protein into relies on nondiscriminative bulk modifi-with previous mass-spectrometry analysis the degradation chamber. This requires ad-cation of proteins, rather than single long on cyclin B ( 9), a protein involved in push-enosine 5′-triphosphate (ATP). Locking the chains, may rationalize many findings at ing the cell through the mitosis phase of proteasome in an ATP-bound state improved odds with prior models ( 15), and suggests the cell cycle, but raises questions about residence times for substrates modified with that single-chain ubiquitination events whether a such modified protein is a good chains of ubiquitin, suggesting that in this could be repurposed for alternative and state, the proteasome exposes a chain recep-nondegradative processes. ■

other study by Lu et al. (5) compares sub-tor near the entry channel.

strates modified with four monoubiquitins, The proteasome does not degrade ubiq-REFERENCES

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with two diubiquitins is the superior prote-( 1). The deubiquitinase Rpn11 is located 4. Y. Lu, W. Weiping, M. W. Kirschner, Science 348, 1248737 asome substrate. This overturns a paradigm near the entry channel (11, 14). Closing (2015).

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with the number of ubiquitins on a sub-has been suggested recently ( 13, 14). 13. E. J. Worden et al., Nat. Struct. Mol. Biol. 21, 220 (2014). strate, Lu et al. (5) reveal cooperative bind-The findings of Lu et al. (4, 5) reveal how 14. G. R. Pathare et al., Proc. Natl. Acad. Sci. U.S.A. 111, 2984 ing for the first three ubiquitin molecules, protein degradation can be used as a rapid (2014).

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time with additional ubiquitin. Although cesses. The proteasome has a quantitative 10.1126/science.aab0931184 10 APRIL 2015 ? VOL 348 ISSUE http://wendang.chazidian.com SCIENCE

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