Location, location, location

Subcellular localization imparts important time and space constraints on signaling pathways. The lipid composition of the different subcellular membranes targets proteins to specific locations in the cell. A well-studied example of spatial organization involves the Ras GTPase isoforms Hras, Nras, and Kras4B. All three isoforms are farnesylated and Nras and Hras are singly and doubly palmitoylated, respectively. Lipid modification of these proteins determines their localization, with Kras found only at the plasma membrane (PM) and Nras and Hras found at both the plasma and Golgi membranes. These different Ras subcellular populations have been shown to participate in distinct signaling pathways dependent on compartmentalization. While it is apparent that palmitoylation of Ras isoforms promotes Golgi localization, it is unclear how this localization is maintained, given the frequency of membrane fusion and spontaneous mixing. Using FRAP to study fluorescently–tagged Ras, Bastiaens and colleagues show that the palmitoyl moiety of Hras and Nras undergoes cycles of cleavage and re-attachment to the proteins in different subcellular locations. This acylation cycle regulates Ras association with the PM and Golgi membranes. The data suggest that the degree of palmitoylation as well as the stability of the palmitoylation site could affect the rate of Golgi localization, which can affect activation patterns of the Ras isoforms. In addition, the de-/re-acylation cycle occurs on both N- and C-terminal palmitoylated peptide sequences. The authors propose that the Ras acylation cycle may be a universal mechanism used to maintain peripheral membrane protein localization. (Science, published online 11 February 2005, 10.1126/science.1105654) MM

A helicase meets p53

DEAD-box helicases use the energy from ATP hydrolysis to rearrange RNA structures or remodel RNA–protein complexes. In addition to their roles in different RNA metabolic pathways, some RNA helicases, like p68, also function in transcription regulation and have been implicated in tumor development and regulation of cell growth. Recent studies have shown that the increased expression of p68, together with the presence of heavily poly-ubiquitylated forms and loss of normal p68, are early events in the development of colon carcinomas. How could the modification or loss of an RNA helicase affect cell growth? Fuller-Pace and colleagues find that p68 functions as a transcriptional coactivator of p53, a well–characterized tumor suppressor. In normal cells, p53 controls cell growth and senescence as well as mediates apoptosis in response to stress such as DNA damage and aberrant oncogene expression. The authors show that p68 interacts with p53 and stimulates transcription from p53-responsive promoters for genes involved in apoptosis and growth arrest. They also show that RNAi suppression of p68 inhibits p53 target gene expression in response to DNA damage, as well as p53-dependent apoptosis. These data indicate that p68 is important for the p53 transcriptional response to DNA damage and define a new mechanism by which p53 function is regulated. How p68 and p53 cooperate to suppress tumor growth is not known. The authors suggest that since p68 interacts directly with p53 and with components of the transcription machinery, the helicase may assist p53 in its association with the transcription initiation complex at target promoters. In so doing, p68 may function as a tumor cosuppressor. (EMBO J. 24, 543–553, 2005) EJ

Caps off

Gene expression in eukaryotes is regulated in part by mRNA degradation. Two major pathways regulate the turnover of mRNA in the cell. In both cases the first step is the shortening of the poly(A) tail (deadenylation) at the 3′ end of the mRNA. In the 5′→3′ pathway, deadenylation is followed by removal of the mRNA cap that exposes the 5′ end of the mRNA for exonucleolytic degradation. In the 3′→5′ pathway, degradation of the mRNA continues from the 3′ end following deadenylation by the cytoplasmic exosome. Decapping is a control point in the 5′→3′ decay pathway and mRNAs differ in their mechanism of decapping both with regard to the requirement for deadenylation and the role of trans-acting factors. Two related proteins, Edc1p and Edc2p, have been previously identified as enhancers of decapping. How might the mRNAs for these decapping enhancers themselves be regulated? Muhlrad and Parker examined the mechanism of degradation of EDC1 mRNA and found that it is not deadenylated but is decapped. Moreover, they show that a poly(U)-rich region of the EDC1 3′ UTR protects the mRNA from deadenylation and is required for deadenylation-independent decapping. Since the EDC1 mRNA bypasses the requirement for deadenylation, its level in the cell would be sensitive to the decapping rates. In addition, because Edc1p enhances decapping, decreases in the decapping rate would preferentially stabilize the EDC1 mRNA and produce more Edc1p, which would increase the rate of decapping. These findings illustrate the different ways decapping can be regulated and identify a possible regulatory feedback loop. The authors also show that the rate of EDC1 mRNA decapping decreases with the loss of Not2p, Not4p and Not5p that are parts of the deadenylation complex. This suggests a possible link between the mRNA deadenylation and decapping machineries. (EMBO J. advance online publication 10 February 2005, 10.1038/sj.emboj.7600560) BK

The gatekeeper

Proteins enter the ER via a transmembrane pore called the translocon. The transport process is co-translational, in which synthesis of nascent polypeptide chains on the ribosome is coupled to translocation through the translocon. To prevent ion leakage through the pore, the translocon is sealed at the lumen side in the inactive state, either directly or indirectly, by BiP, an Hsp70 protein. BiP is a molecular chaperone with two major activities: substrate binding and ATP hydrolysis. These activities are performed by distinct structural domains in BiP and are regulated by allosteric communication between these domains, as well as by cochaperones such as those containing a conserved J-domain. During translocation, the ribosome engages and seals the translocon from the cytoplasmic side. When the nascent polypeptide chain exiting the ribosome becomes long enough to reach the lumen, the gate on the lumen side opens. To understand how BiP mediates gating of the translocon, Johnson and colleagues reconstituted microsomes containing translocons with BiP mutants that have various functional defects. They then monitored whether these mutants can form functional gates. The data show that sealing the translocon requires ADP bound to BiP, whereas pore opening requires conformational changes in BiP induced by ATP binding. They also show that to affect pore closure BiP must directly interact via its substrate-binding cleft with a translocon-associated membrane protein and via its ATPase domain with a membrane-bound J-domain protein. Further understanding of how BiP gates the translocon will require identification of BiP's binding partners and characterizing how these interactions are regulated by BiP's ATP hydrolysis cycle. (J. Cell Biol. 168, 389–399, 2005). HPF

Research highlights written by Hwa-ping Feng, Evelyn Jabri, Boyana Konforti and Michelle Montoya.