The P124A mutation of SRP14 alters its migration on SDS-PAGE without impacting its function

Abstract

SRP14 is a crucial protein subunit of the signal recognition particle (SRP), a ribonucleoprotein complex essential for co-translational translocation to the endoplasmic reticulum. During our investigation of SRP14 expression across diverse cell lines, we observe variations in its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with some cells exhibiting slower migration and others migrating faster. However, the cause of this phenomenon remains elusive. Our research rules out alternative splicing as the cause and, instead, identifies the presence of a P124A mutation in SRP14 (SRP14P124A) among the faster-migrating variants, while the slower-migrating variants lack this mutation. Subsequent ectopic expression of wild-type SRP14P124 or SRP14WT and SRP14P124A in various cell lines confirms that the P124A mutation indeed leads to faster migration of SRP14. Further mutagenesis analysis shows that the P117A and A121P mutations within the alanine-rich domain at the C-terminus of SRP14 are responsible for migration alterations on SDS-PAGE, whereas mutations outside this domain, such as P39A, Y27F, and T45A, have no such effect. Furthermore, the ectopic expression of SRP14WT and SRP14P124A yields similar outcomes in terms of SRP RNA stability, cell morphology, and cell growth, indicating that SRP14P124A represents a natural variant of SRP14 and retains comparable functionality. In conclusion, the substitution of proline for alanine in the alanine-rich tail of SRP14 results in faster migration on SDS-PAGE, but has little effect on its function.

1. Introduction

The signal recognition particle (SRP) is a ribonucleoprotein complex that plays a critical role in co-translational protein translocation to the endoplasmic reticulum (ER). SRP consists of six proteins (SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72), as well as a single SRP RNA, the 7SL RNA. Notably, the SRP9/SRP14 heterodimer, in conjunction with the 5′ and 3′ termini of 7SL RNA, collaboratively forms what is known as the Alu domain [1].

When the signal sequence emerges from the ribosome, it is captured by SRP, and the elongation of the nascent polypeptide is arrested by the Alu domain. Translation resumes when the ribosome–nascent chain-SRP complex (RNC-SRP) docks with the SRP receptor (SR) on the ER. The function of the Alu domain in arresting translation is believed to extend the time available for RNC-SRP to target the SR, therefore ensuring efficient co-translational translocation [2].

Despite limited primary sequence homology, the crystal structures of SRP9/14 exhibit structural similarity. Both proteins form a three-stranded antiparallel β-sheet stacked against two helices. The six β-sheets of the heterodimer create a highly positively charged concave surface, which serves as the primary Alu RNA-binding site (Figure 1A) [3]. Notably, despite their structural resemblance, the internal loop between the β1 and β2 sheets of SRP14 is absent in SRP9. Although the affinity between Alu RNA and SRP9/14 lacking the internal loop decreases 10-fold, this does not compromise elongation arrest [4]. In contrast to the indispensable internal loop, the pentapeptide KRDKK following K95 of SRP14 is crucial for elongation arrest both in vitro and in mammalian cells [5]. The insertion of as few as two alanine residues after K95 can completely abrogate the arrest of elongation [4]. The basic patches of SRP14 serve as a positively charged platform for interactions with ribosomal RNA.