Signal Recognition Particle Mediates a Transient Elongation Arrest of Preprolactin in Reticulocyte Lysate

Signal recognition particle (SRP) is a ribonucleoprotein that functions in the targeting of ribosomes synthesizing presecretory proteins to the ER. SRP binds to the signal sequence as it emerges from the ribosome, and in wheat germ extracts, arrests further elongation. The translation arrest is released when SRP interacts with its receptor on the ER membrane. We show that the delay of elongation mediated by SRP is not unique to wheat germ translation extracts. Addition of mammalian SRP to reticulocyte lysates resulted in a delay of preprolactin synthesis due to increased ribosome pausing at specific sites on preprolactin mRNA. Addition of canine pancreatic microsomal membranes to reticulocyte lysates resulted in an acceleration of preprolactin synthesis, suggesting that the endogenous SRP present in the reticulocyte lysate also delays synthesis of secretory proteins. S I6NAL recognition particle (SRP) t is an llS ribonucleoprotein particle that facilitates the translocation of nascent secretory proteins across the rough endoplasmic reticulum. The availability of extracts that reproduce secretory protein translocation has made it possible to derive a model for SRP function. According to this model, SRP binds with a low but measurable affinity to all ribosomes. Upon emergence of the signal sequence of a newly translated protein from the ribosome, the affinity of SRP for the ribosome increases by several orders of magnitude. The ribosome-nascent chain-SRP complex is then targeted to the endoplasmic reticulum, where SRP interacts with the SRP receptor (also called docking protein). SRP can thus be thought of as an adapter between the cytoplasmic protein synthetic machinery and the translocation apparatus in the endoplasmic reticulum membrane (reviewed by Walter and Lingappa, 1986; Hortsch and Meyer, 1986). Many of the experiments leading to the above model were performed in a wheat germ translation extract that was supplemented with canine SRP and canine rough microsomes. If SRP is added to these extracts in the absence of microsomal membranes, SRP arrests or retards translation of the nascent polypeptide chain (Walter and Blobel, 1981; Ibrahimi, 1987; Rapoport et al., 1987). This "elongation arrest" occurs after the nascent chain is at least 70 amino acids long. As 30-40 amino acids of the growing polypeptide chain are sequestered within the ribosome, this corresponds to the length at which the signal sequence has emerged completely from the ribosome. For certain proteins, a discrete "arrested fragment" can be detected (Walter and Blobel, 1981; Meyer 1. Abbreviations used in this paper: 7raG, 7-methylguanosine-5'-monophosphate; SRP, signal recognition particle. et al., 1982), while for other proteins, SRP arrest occurs at multiple sites during peptide elongation (Lipp et al., 1987). An SRP-mediated delay in the synthesis of secretory proteins could serve at least two distinct functions. First, a pause in polypeptide elongation would increase the length of time that the nascent polypeptide remains in an unfolded conformation compatible with efficient translocation. Second, an elongation arrest could provide a regulatory mechanism by which cells rapidly modeulate the synthesis of secretory proteins in response to environmental changes. Because the ability of SRP to delay the translation of secretory proteins has important functional implications, it is necessary to demonstrate that this phenomenon is not unique to a wheat germ translation system supplemented with canine SRP. Several attempts to demonstrate an SRP-mediated translation arrest in other systems have been unsuccessful. Meyer (1985) found that the addition of even very high amounts of canine SRP had no effect on the synthesis of a secretory protein in either rabbit reticulocyte or HeLa cell lysates. Also, fractions containing either maize or wheat germ SRP did not arrest translation when added to wheat germ extracts (Prehn et al., 1987; Campos et al., 1988). Thus, the ability of SRP to arrest translation has been called into question as an artifact of an in vitro system reconstituted from heterologous components. We have reinvestigated the effects of SRP on translation of secretory proteins in the rabbit reticulocyte lysate. We demonstrate that the addition of even low amounts of canine SRP causes a measurable delay in polypeptide elongation. Our results also indicate that the endogenous SRP present in the reticulocyte lysate specifically slows elongation of nascent