We have characterized the tsr1-1 allele as a suppressor of the thermosensitive growth phenotype of the 7S RNA mutation scr2.II-13 which affects the stability of the signal recognition particle (SRP) in the yeast Yarrowia lipolytica. Native immunoprecipitation experiments using anti-Srp19p sera showed that tsr1-1 ensures a complete recovery of the stability of the SRP. In a wild type SCR2+ context, the tsr1-1 allele confers growth defect at 34°C. Study of the synthesis and secretion of two secretory reporter proteins by pulse chase labeling and immunoprecipitation showed that the level of precursors in the tsr1-1(SCR2+) strain is /70% less than in the wild type strain whereas global protein synthesis is practically unaffected. We have cloned the TSR1 gene by complementing the thermosensitive phenotype of a tsr1-1(SCR2+) mutant. Analysis of the TSR1 DNA sequence revealed an ORF of 1383 base pairs, encoding a serine-rich protein of 461 amino acids with an amino terminal signal peptide, and a membrane spanning domain of 20 amino acids. Protease protection assays confirmed that Tsr1p was a membrane spanning protein. Disruption of the TSR1 revealed that it is an essential single copy gene. Homologues of the TSR1 gene were identified in S. cerevisiae (YHC8) and H. polymorpha (YLU2). S. cerevisiae yhc8::Kanr disruptants were viable, but accumulated precursors of the CPY reporter molecule. We propose that Tsr1p, and its homologue Yhc8p in S. cerevisiae, are both ER membrane proteins and could play an important role in the translocation pathway of secretory proteins.