p97 Adaptor Choice Regulates Organelle Biogenesis

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and SA1 subunits of cohesin, and suggest that Wapl might negatively regulate the acitivity of this subcomplex to prevent cohesin from reassociating with the chromosomes following release. Further experiments are needed to determine the in vivo relevance of these different interactions. Both studies suggest that Wapl works to drive cohesin off of the chromatin. The slower dynamics of the cohesin-DNA interaction and the failure to resolve sister chromatids in mitosis in cells lacking Wapl suggest that Wapl can affect the level of cohesin that has ‘‘established’’ cohesion, as well as that which is only ‘‘chromatin associated.’’ It has been difficult to understand the difference between these two classes of cohesin. The identification of a regulatory protein that is able to increase the level of established cohesion may provide an important tool for improving our understanding of the molecular nature of this distinction. Figure 1. Wapl and Cohesin Dynamics Cells with reduced levels of Wapl show changes in cohesin localization and dynamics. In interphase nuclei, (green ovals, left) there is an overall increase in the steady state levels of chromatin-associated cohesin (blue dots), and the residence time of the dynamic pool of cohesin is more than doubled from 8 to 18 min. In mitotic cells (right), there is an increase in the amount of arm-associated cohesin on the condensed chromosomes, and this corresponds to a failure to resolve sister chromatids in metaphase. Anaphase separation of sister chromatids is delayed but appears normal.

an increase in levels of mitotic cohesion? Kueng et al. (2006) show that in interphase there is an overall increase in chromatin-associated cohesin, and that the residence time of the ‘‘fast’’ pool of cohesin is more than doubled. For technical reasons it is difficult to determine residence time for the stably bound cohesin in G2, but the authors speculate that Wapl might similarly affect the dynamics of this pool of cohesin as well. What might Wapl be doing? The two groups have arrived at different conclusions about the way in which Wapl interacts with the cohesin complex. Kueng et al. show that Wapl forms a subcomplex with the Pds5 subunit of cohesin, a weakly associated subunit with seemingly paradoxical properties (Losada et al., 2005). They suggest that Pds5 and Wapl might work in opposition to each other to regulate the association of cohesin with chromosomes. In contrast, Gandhi et al. show that Wapl can form a stable subcomplex with the Scc1

Developmental Cell 11, December, 2006 ª2006 Elsevier Inc.

Susannah Rankin1 1 Molecular, Cell, and Developmental Biology Research Program Oklahoma Medical Research Foundation 825 N.E. 13th Street Oklahoma City, Oklahoma 73104 Selected Reading Dobie, K.W., Kennedy, C.D., Velasco, V.M., McGrath, T.L., Weko, J., Patterson, R.W., and Karpen, G.H. (2001). Genetics 157, 1623–1637. Gandhi, R., Gillespie, P., and Hirano, T. (2006). Curr. Biol., in press. Published online November 16, 2006. 10.1016/j.cub.2006.10.026. Gerlich, D., Koch, B., Dupeux, F., Peters, J.M., and Ellenberg, J. (2006). Curr. Biol. 16, 1571–1578. Ivanov, D., and Nasmyth, K. (2005). Cell 122, 849–860. Kueng, S., Hegemann, B., Peters, B.H., Lipp, J.J., Schleiffer, A., Mechtler, K., and Peters, J.M. (2006). Cell 127, 955–967. Lengronne, A., Katou, Y., Mori, S., Yokobayashi, S., Kelly, G.P., Itoh, T., Watanabe, Y., Shirahige, K., and Uhlmann, F. (2004). Nature 430, 573–578. Losada, A., Yokochi, T., and Hirano, T. (2005). J. Cell Sci. 118, 2133– 2141. Oikawa, K., Ohbayashi, T., Kiyono, T., Nishi, H., Isaka, K., Umezawa, A., Kuroda, M., and Mukai, K. (2004). Cancer Res. 64, 3545–3549. Uhlmann, F. (2003). Curr. Biol. 13, R104–R114. Verni, F., Gandhi, R., Goldberg, M.L., and Gatti, M. (2000). Genetics 154, 1693–1710.

DOI 10.1016/j.devcel.2006.11.005

p97 Adaptor Choice Regulates Organelle Biogenesis The AAA protein p97 requires adaptor-like cofactors for its numerous cellular functions. In this issue of Developmental Cell, Uchiyama et al. (2006) identify p37 as a p97 adaptor that is required constitutively

for ER and Golgi membrane fusion, analogous to the mitotic membrane fusion role of the adaptor p47. Their study suggests that related p97 adaptors involved in similar cellular pathways can be subject to differential regulation. The AAA ATPase p97 has a remarkable spectrum of cellular functions, most of which relate to the ubiquitin

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system (Halawani and Latterich, 2006). Many key cellular pathways, such as cell division, ER-associated degradation (ERAD), cell proliferation, apoptotic switches, organelle assembly, and the clearance of nuclear and cytosolic protein aggregates, depend on p97 for proper function. Although the precise biochemical role of p97 is poorly understood, the function of adaptor proteins that target and connect p97 to distinct cellular pathways is becoming increasingly clear (Dreveny et al., 2004). p97 adaptors can be classified into three major classes based on their ternary interactions and p97 binding domains. These classes are the bipartite class (two interacting proteins make up the adaptor), the UBX (ubiquitin interaction domain X)-domain-containing class, and the novel interaction-domain-containing class. One of the best-characterized p97 adaptors is the bipartite Ufd1Npl4 complex, which links p97 to ubiquitin-dependent protein degradation events, such as ERAD. Npl4 binds to polyubiquitinylated substrates and links to p97’s N terminus via Ufd1, which binds both to Npl4 and p97 (Meyer et al., 2002). UBX domain adaptors such as p47 are characterized by the presence of a UBX domain, which mediates their binding to the N terminus of p97. p47 was identified as a p97-interacting protein needed for the homotypic fusion of Golgi fragments (Kondo et al., 1997). In addition to its UBX domain, p47 contains a UBA domain which facilitates its binding to monoubiquitinylated substrates (Meyer et al., 2002). Closer examination of p47-function revealed that it is essential for cell-cycle-dependent fusion of Golgi and ER fragments (Kano et al., 2005; Uchiyama and Kondo, 2005). However, despite earlier assumptions, it does not seem to be required for constitutive homotypic fusion of ER fragments and Golgi fragments, raising the question of whether p97 function in interphase organelle biogenesis is related to its mitotic regulation. In their new study published in this issue of Developmental Cell, Uchiyama et al. (2006) show that both mitotic and constitutive ER and Golgi assembly require p97. Furthermore, they show that rather than involving the p47 adaptor, constitutive fusion of these membranes requires a newly identified adaptor, p37, which acts in conjunction with p97 and which also has a UBX domain to facilitate p97 binding. The two adaptor-p97 complexes are mutually exclusive (i.e., there are no p97 hexamers consisting of hybrid p47/p37 adaptors), suggesting that each assembled complex has a dedicated cellular function. Unlike p47, which has a monoubiquitin interacting UBA domain, p37 lacks an ubiquitin-binding domain, suggesting that the binding substrates may be significantly different. Additional information also supports the idea that there are fundamental differences between p47 and p37 adaptor function. Previous data showed that the p47/p97 complex functions together with the deubiquitinylating protease VCIP135 (Uchiyama et al., 2002) and that the deubiquitinylating activity is required to activate Golgi and ER membranes and enable them to fuse. However, even though p37/p97 is also associated with VCIP135, the latter’s deubiquitinylating activity is dispensable for membrane fusion function. One possible explanation for this observation could be that the constitutive pathway involves the extraction and subsequent degradation of a substrate, such as a fusion inhibitor,

while the mitotically regulated pathway may recycle a component that is subject to repeated ubiquitinylation and deubiquitinylating cycles. The VCIP135 requirement may merely be structural in the p37/p97 complex. Another distinction between the two pathways is that while p47/p97 binds to syntaxin5, the p37/p97 complex requires the tethering components p115-GM1130 and GS12. Thus, it seems increasingly likely that p97-dependent organelle assembly during mitosis and in interphase are fundamentally different processes. Uchiyama et al.’s findings also suggest that a major role of p97 adaptors in membrane fusion is to target p97 to appropriate cellular membranes and organelle fusion machineries. However, instead of targeting to different organelles, it seems that different adaptors are subject to different types of regulation (mitotic versus constitutive), and may even target distinct organelle fusion complexes on the ER and Golgi membranes. If this is the case, then the p97 and adaptor requirement in fusion pathways would not be to determine the specificity of membrane fusion, but instead would represent a common component of otherwise distinct fusion machineries. We know from previous studies that differential phosphorylation of p47 by cdc2 kinase determines when p47 is available for its role in organelle membrane fusion and ER exit site assembly (Kano et al., 2004). p37, on the other hand is readily available for ER and Golgi membrane fusion throughout the cell cycle. Thus, the cell may have two distinct requirements for its organelle assembly complexes—a constitutive requirement to remedy spontaneous fragmentation of organelles and ensure that they remain intact throughout the cell cycle, and a mitotically regulated requirement to during mitosis, where temporally regulated fragmentation is needed to ensure correct partitioning of organelles such as the Golgi apparatus. Although the current study answers a key question related to p97’s role in membrane fusion and distinguishes between its roles in constitutive and mitotic fusion, it raises many more questions that relate to p97 function. For example, what are the actual p97 substrates, and what is the role of the adaptors? Are p97 and associated proteins part of an elaborate ubiquitinylation-deubiquitinylating complex (Halawani and Latterich, 2006)? If so, what are the corresponding E3 ligases and the target proteins? At a more fundamental level, this new study does not really bring us any closer to understanding how p97 actually functions in membrane fusion, and this is obviously also a key question. In vitro reconstitution of these pathways followed by detailed mechanistic and structural analysis should help address some of these important issues. Martin Latterich1 1 Department of Anatomy and Cell Biology McGill University 3640 University Street Montreal, Quebec, H3A 2B2 Canada Selected Reading Dreveny, I., Pye, V.E., Beuron, F., Briggs, L.C., Isaacson, R.L., Matthews, S.J., McKeown, C., Yuan, X., Zhang, X., and Freemont, P.S. (2004). Biochem. Soc. Trans. 32, 715–720.

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Halawani, D., and Latterich, M. (2006). Mol. Cell 22, 713–717.

Meyer, H.H., Wang, Y., and Warren, G. (2002). EMBO J. 21, 5645–5652.

Kano, F., Tanaka, A.R., Yamauchi, S., Kondo, H., and Murata, M. (2004). Mol. Biol. Cell 15, 4289–4298. Kano, F., Kondo, H., Yamamoto, A., Kaneko, Y., Uchiyama, K., Hosokawa, N., Nagata, K., and Murata, M. (2005). Genes Cells 10, 989–999. Kondo, H., Rabouille, C., Newman, R., Levine, T.P., Pappin, D., Freemont, P., and Warren, G. (1997). Nature 388, 75–78.

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Uchiyama, K., and Kondo, H. (2005). J. Biochem. (Tokyo) 137, 115–119. Uchiyama, K., Jokitalo, E., Kano, F., Murata, M., Zhang, X., Canas, B., Newman, R., Rabouille, C., Pappin, D., Freemont, P., et al. (2002). J. Cell Biol. 159, 855–866. Uchiyama, K., Totsukawa, G., Puhka, M., Kaneko, Y., Jokitalo, E., Dreveny, I., Beuron, F., Zhang, X., Freemont, P., and Kondo, H. (2006). Dev. Cell 11, this issue, 803–816.

DOI 10.1016/j.devcel.2006.11.004

Revenge of the NRD: Preferential Degradation of Nonfunctional Eukaryotic rRNA Eukaryotic cells have several quality control systems that monitor the proper processing of RNAs during RNA biogenesis or the function of cytoplasmic mRNAs. A recent study in Molecular Cell by LaRiviere et al. (2006) shows that after production, mature rRNA is also subject to quality control and nonfunctional ribosomes are targeted for destruction by a novel ribosome surveillance mechanism. An essential process to life is the production of the protein synthesis machinery, including ribosomes, and the mRNAs it translates. In eukaryotic cells, the biogenesis of functional RNAs, such as rRNA, tRNA, and snRNAs, as well as mRNAs involves multiple RNA processing events and several orchestrated rearrangements of RNA-protein complexes that catalyze and promote sequential biogenic steps, ultimately leading to cytoplasmic function. Given the complexity of the process of RNA biogenesis and function, it is reassuring that eukaryotic cells have evolved multiple classes of quality control systems that degrade aberrant RNAs. One class of quality control occurs during biogenesis, such that RNAs with defects in RNA processing, or RNP assembly, are subject to degradation. For example, eukaryotic cells contain specialized nuclear poly(A) polymerases that adenylate a variety of defective RNAs including hypomethylated and unprocessed tRNAs, mutant snRNAs, and defective pre-rRNAs, which then leads to their degradation by the nuclear exosome (Fasken and Corbett, 2005; Kadaba et al., 2006). Similarly, defective pre-mRNAs with improper polyadenylation can be targeted for retention and/or degradation by the nuclear exosome (Fasken and Corbett, 2005). Additional systems to assess proper RNA biogenesis include a role for the conserved protein Ro in quality control by binding misfolded RNAs such as pre-rRNAs (Stein et al., 2005). A second class of quality control ensures that the functional competence of the transcript is assessed and nonfunctional transcripts are degraded. Three distinct

functional quality controls have been identified that assess the translational competence of cytoplasmic mRNAs. Specifically, mRNAs with premature translational termination codons (nonsense-mediated decay [NMD]), no translation termination codon (nonstop decay pathway [NSD]), or strong stalls to translation elongation (no-go decay [NGD]) are recognized and subject to increased rates of degradation (Fasken and Corbett, 2005; Doma and Parker, 2006). In each case, these defective mRNAs are recognized due to defects in the normal process, or rate, of translation. In a new study from Melissa Moore’s group, LaRiviere and colleagues show that rRNA that is part of mature eukaryotic ribosomes is subject to functional quality control, in a novel surveillance pathway referred to as nonfunctional rRNA decay (NRD) (LaRiviere et al., 2006). Together, the above studies suggest that many, if not all, RNA transcripts are subject to extensive surveillance mechanisms at every step of their biogenesis and function. To investigate if eukaryotes have a quality control mechanism to assess rRNA functionality, the authors expressed yeast rRNAs containing point mutations in either the peptidyl transferase center (PTC) of the 25S rRNA or the decoding site (DCS) of the 18S rRNA. These mutations are analogous to bacterial rRNA mutations shown to inhibit ribosome function in vitro (Youngman et al., 2004) and to be dominant-lethal when overexpressed in vivo (Green et al., 1997). Consistent with these mutations inhibiting ribosome function in yeast as well, the 25S rRNA mutants or 18S rRNA mutants were unable to support growth in the absence of endogenous rRNAs. However, in contrast to the dominant lethality seen in bacteria, the expression of the mutant rRNAs together with endogenous rRNAs had no deleterious effects in yeast, which suggested that yeast cells may possess one or more systems to prevent nonfunctional ribosomes from interfering with translation. LaRiviere et al. (2006) present several lines of evidence that the nonfunctional rRNA is preferentially degraded by a process they term nonfunctional rRNA decay (NRD). The key observation is that direct measurements of rRNA decay rates showed that nonfunctional mutant 18S and 25S rRNAs were degraded much faster than wild-type rRNAs, which leads to a 5- to 10fold reduction in their steady-state levels. Moreover,