Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane.

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.

A second signal recognition event required for translocation into the endoplasmic reticulum.

As summarized in this minireview, two different signal recognition events, one involving SRP and the other involving proteoliposomes containing the Sec61p complex, have been identified. In cotranslational protein transport, it seems that both recognition events are required for efficient translocation of the protein into the lumen of the ER. The requirement for SRP can, under certain experimental conditions, be circumvented by depletion of NAC, a heterodimeric complex that can block the tight association of nascent chain-ribosome complexes to the Sec61p complex in the ER membrane. In posttranslational protein transport, the Sec61p complex contains additional protein subunits that are required for function. It should be noted that, in all the experiments performed in which the role of SRP in cotranslational protein translocation is circumvented (Jungnickel and Rapoport, 1995; Lauring et al., 1995a, 1995b), stable translocation intermediates are allowed many minutes to establish productive interactions with the membrane. In contrast, during conditions in which the nascent chain can elongate (e.g., in vivo), the nascent chain-ribosome complex only has a brief time window during which it can initiate translocation (reviewed by Walter and Johnson, 1994). It is possible that, under these conditions, productive translocation even in the absence of NAC would require SRP. The isolation of NAC-deficient extracts that support protein synthesis will allow a test of this possibility. Finally, the role that lipids themselves may play in protein transport should not be ignored. Gierasch and coworkers (Hoyt and Gierasch, 1991, and references therein) have shown that bacterial signal peptides have an intrinsic ability to interact with lipid and that the relative ability of a mutant signal sequence to interact with lipid correlates with its function as a signal sequence in vivo. Thus, the signal sequence-discriminatory role defined by Jungnickel and Rapoport (1995) may in fact be played by lipid, with the Sec61p complex playing a necessary but nondiscriminatory role in the process. In this light, it is interesting that Martoglio et al. (1995) recently demonstrated that the signal sequence of preprolactin could be cross-linked to phospholipid. Analysis of the cross-linking efficiency of the signal sequence to phospholipid at different nascent chain lengths and with mutant signal sequences will help define the role that phospholipid plays in the process.

Binding sites of the 19-kDa and 68/72-kDa signal recognition particle (SRP) proteins on SRP RNA as determined in protein-RNA "footprinting".

We have used the nuclease alpha-sarcin to map the binding sites of the 19-kDa and the 68/72-kDa proteins of signal recognition particle (SRP) on SRP RNA. We found that the regions of protection to nuclease afforded by the two proteins were distinct. p19 protected primarily the two tips in the RNA secondary structure. p68/72 protected a large region extending across the center of the particle and altered the nuclease pattern in the regions that p19 would bind, suggesting that these two proteins may be in close proximity in the particle. The protection afforded by the two proteins in combination was equal to the sum of the individual protections. We have not observed cooperativity in the binding of these two proteins as assessed by the protection assay; nor do we have any evidence that the structure becomes more compact as it assembles. The map derived from this "footprint" analysis places the signal recognition domain (p54 bound to the RNA via the 19-kDa protein) and the elongation arrest domain (associated with the Alu end of the particle) on opposite ends of the particle. Thus, it is possible that SRP recognizes signals by the direct interaction of p54 with the signal sequence at the nascent chain exit site and simultaneously blocks elongation by the entrance of p9/14 into the aminoacyl tRNA site 16 nm away.

Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: analysis of biochemical mutants of SRP.

Signal recognition particle (SRP), a small ribonucleoprotein required for targeting secretory proteins to the ER, has three known functions: signal recognition, elongation arrest, and translocation promotion. Because SRP is inactivated by the sulfhydryl alkylating reagent N-ethylmaleimide (NEM), we have attempted to establish structure-function relationships within SRP by assembling particles in which a single protein is modified. Alkylation of the 68/72 kd protein of SRP yields a particle that arrests elongation but fails to promote translocation and no longer interacts with SRP receptor. Alkylation of the 54 kd protein yields a particle that fails to recognize signal sequences. This approach has allowed us to map activities to specific protein domains on SRP, and should be generally useful for analyzing other ribonucleoproteins.

Removal of the Alu structural domain from signal recognition particle leaves its protein translocation activity intact.

Alu-like elements comprise the most abundant family of interspersed repetitive sequences in primates and rodents, and contain many features of processed genes, suggesting that they were initially derived by reverse transcription of processed RNA transcripts. Transcripts containing Alu family members are represented in heterologous nuclear RNAs, cytoplasmic messenger RNAs and small RNAs, although nothing is known about their function. Evolutionary studies strongly suggest that the parent RNA for the Alu-like elements is the highly conserved 7SL RNA, which is an essential component of signal recognition particle (SRP), a small cytoplasmic ribonucleoprotein whose function is the targeting of nascent secretory and membrane proteins to the rough endoplasmic reticulum (for a review see ref. 6). 7SL RNA is composed of both unique and Alu-like sequences. SRP is rod-shaped and, in addition to its RNA, contains four proteins (two monomers composed of a polypeptide of relative molecular mass (Mr) 19,000 (19K) and one of 54K, and two heterodimers, one composed of a 9K and a 14K polypeptide, and the other composed of a 68K and a 72K polypeptide, respectively). The RNA moiety is required for SRP activity, as well as for structural integrity of the particle. To investigate whether the Alu-like segments of 7SL RNA have a specific role in SRP activity, we have now purified and analysed a SRP subparticle that is created upon extensive digestion with micrococcal nuclease and entirely lacks the Alu-like sequences. We find that it contains the 72/68K, 54K and 19K proteins tightly bound, but lacks the 9/14K protein. In vitro activity assays demonstrated that the subparticle could still promote secretory protein translocation across the microsomal membrane, but could no longer trigger an arrest of pre-secretory protein synthesis. Re-addition of the 9/14K protein did not restore the elongation arrest. We conclude that the region of SRP comprised of the Alu-like RNA and the 9/14K protein exists in a distinct structural domain which is not required for the protein translocation promoted by SRP but apparently confers elongation-arresting activity on the particle.

The affinity of signal recognition particle for presecretory proteins is dependent on nascent chain length.

We have developed an assay in which incomplete preprolactin chains of varying lengths are targeted to the endoplasmic reticulum (ER) membrane in an elongation independent manner. The reaction had the same molecular requirements as nascent chain translocation across the ER membrane, namely, it was signal recognition particle (SRP) dependent, and required the nascent chain to be present as peptidyl tRNA (i.e. most likely ribosome associated) and to have its signal sequence exposed outside the ribosome. We found that the efficiency of the targeting reaction dropped dramatically as the chains grew longer than 140 amino acids in length, which probably reflected a decrease in affinity of the nascent chain-ribosome complex for SRP. Thus at physiological SRP concentrations (10 nM) there appears a sharp cut-off point in the ability of these chains to be targeted, while at high SRP concentrations (270 nM) all chains could be targeted. In kinetic experiments, high concentrations of SRP were found to change the time in elongation after which translocation of the nascent polypeptide could no longer occur.

pipsqueak encodes a novel nuclear protein required downstream of seven-up for the development of photoreceptors R3 and R4.

Photoreceptor induction in the Drosophila eye is mediated by activation of the Ras signal transduction cascade. Although this process is well understood, little is known about how the diversity of photoreceptor subtypes is generated. The pipsqueak (psq) gene is expressed at high levels in the R3/R4 precursors during eye development and this expression depends on seven-up (svp) gene function. Moreover, strong psq alleles are dominant suppressors of a svp-induced cone cell transformation phenotype. Although the gene was previously identified and described as a member of the maternal posterior group of genes, the strong semilethal alleles isolated here demonstrate a specific requirement for psq function downstream of svp for the development of photoreceptors R3/R4. The gene has three independent 5' ends and codes for several nuclear protein isoforms, some containing the POZ domain which has been implicated in protein-protein interactions. Interestingly, all viable alleles with a maternal posterior group phenotype cluster around one specific 5' exon, while all semilethal alleles have lesions which map to a different alternative 5' exon.

Small ribonucleoproteins in Schizosaccharomyces pombe and Yarrowia lipolytica homologous to signal recognition particle.

We have partially purified ribonucleoproteins (RNPs) from Schizosaccharomyces pombe and Yarrowia lipolytica with properties resembling those of mammalian signal recognition particle (SRP). In both species of yeast we have identified a single major RNA species in the size range of SRP RNA (256 nucleotides in S. pombe and 270 nucleotides in Y. lipolytica) present in postribosomal salt extracts of the cytoplasm. The RNPs containing these RNAs sediment in sucrose gradients at 11 S and 10 S for S. pombe and Y. lipolytica, respectively. Analysis of genomic clones of these RNAs has revealed that (i) they are encoded by single copy genes; (ii) they share two short conserved sequences that match the A and B boxes defined for polymerase III promoters; (iii) they can be folded into secondary structures that closely match that defined by phylogenetic analysis of higher eukaryotic SRP RNAs; and (iv) they show primary sequence conservation in short regions predicted to be single stranded. Both of the yeast RNAs bind under stringent conditions to canine SRP proteins. Most importantly, RNase protection of the S. pombe RNA by the individual canine SRP proteins, p19 and p68/72, shows that the proteins recognize homologous elements of the mammalian and yeast RNA. Taken together these data suggest strongly that we have identified yeast SRP homologues.

Autonomous determination of anterior structures in the early Drosophila embryo by the bicoid morphogen.

A small number of maternal effect genes determine anterior-posterior pattern in the Drosophila embryo. Embryos from females mutant for the maternal gene bicoid lack head and thorax. bcd mRNA becomes localized to the anterior tip of the egg during oogenesis and is the source for the morphogen gradient of bcd protein. Here we show that in vitro transcribed bicoid mRNA that has its own leader sequences substituted by the Xenopus beta-globin 5' untranslated sequences is translated more efficiently than bicoid mRNA with the natural 5' mRNA leader when tested in vitro and in Drosophila Schneider cells. When injected into bicoid mutant embryos, only the bcd mRNA with the beta-globin leader sequence, substituted for the natural leader, is able to induce anterior development. We used P-transformation to show that sequences in the 5' leader are neither necessary for localization of the transcript nor for the translational block of the bcd mRNA during oogenesis. For our injection experiments, we used only one of the identified splicing forms of bcd mRNA. The bcd protein species derived from this mRNA is able to induce anterior development at any position along the anterior-posterior axis. Thus bicoid protein can induce development of head and thorax independent of any other specifically localized morphogenetic factor. Our findings further support the notion that the concentration gradient of bcd protein, and not the existence of different forms of bcd protein, is responsible for specifying subregions of the embryo.

pipsqueak, an early acting member of the posterior group of genes, affects vasa level and germ cell-somatic cell interaction in the developing egg chamber.

We have identified a new member of the posterior group of genes, which we call pipsqueak. We show that pipsqueak acts after the establishment of the oskar posterior anchor but before the localization of vasa protein during oogenesis. Characterization of multiple alleles at the pipsqueak locus shows that pipsqueak, like vasa, is required for early stages of oogenesis, including but not limited to formation of the egg chamber and progression through Stage 6 of oogenesis. Genetic interaction studies suggest that pipsqueak acts at least partially through vasa; molecular studies indicate that pipsqueak affects vasa level in the ovary. We compare vasa and pipsqueak mutant phenotypes in order to determine whether pipsqueak acts solely through vasa, and present a model for the role of pipsqueak in posterior pattern formation.