Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and kifodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K-*52K-*32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preprol-proII-proIlI amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.

Abstract. Replacement of the signal recognition particle (SRP) 7S gene (SCRI) on a replicating plasmid with scrll (G to A at 129 and A to T at 131 in the consensus sequence -GNAR-in the loop of domain III) resulted in temperature sensitivity for growth of cells in which both chromosomal SRP 7S RNA genes were deleted . Pulse-chase immunoprecipitation experiments were done after a shift to non-permissive temperature using the major secreted protein the alkaline extracellular protease (AEP) as a reporter molecule. No untranslocated AEP precursor was detected in a strain with scrll on a plasmid, but the amount of the largest AEP precursor (55 kD) immunoprecipitated as a percentage of total protein synthesized was reduced 68% compared to an isogenic strain with SCRI N higher eukaryotes, evidence suggests that the signal recognition particle (SRP)' is essential for protein translocation across the ER membrane. SRP is a soluble IIS ribonucleoprotein composed of six polypeptides (72,68,54,19,14, and 9 kD) and a single 7S RNA of 300 nucleotides (66,74) . From the study of in vitro systems, a model has been proposed in which SRP functions as an adapter between the translational machinery in the cytoplasm and the translocational machinery in the ER membrane (49,66,74) . The functions of individual SRP proteins have been elucidated by the study of "mutant" SRPs reconstituted in vitro (63, 65) . Three functions ofSRP have been identified : signal recognition, elongation arrest, and translocation promotion (66). Translation of mRNAs coding for secretory proteins begins on free ribosomes in the cytoplasm . When the polypeptide chain has elongated sufficiently, SRP binds the signal sequence/ribosome complex . This interaction results in arrest or pausing of translation (74, 78) . The SRP/ribosome complex is then targeted to the SRP receptor, an ER integral membrane protein. This results in the release of SRP and translational arrest, and the polypeptide is then co-translationally translocated into the ER. During or shortly after on the plasmid . The possibility that an untranslocated precursor was synthesized but not detected because of instability was largely eliminated by detection of a 53-kD untranslocated precursor of a mutated AEP (P17M; methionine replaced proline in the second position of the pro-peptide) which chased to the 55-kD translocated AEP precursor. Thus, SRP has a role in the biosynthesis of AEP. Possibly, the scrll mutation does not affect signal recognition or translational arrest but instead results in maintenance of translational arrest of AEP synthesis . The results also suggest that AEP can be translocated in vivo either co-translationally in which SRP is at least involved in biosynthesis or posttranslationally without SRP involvement .translocation, signal peptide cleavage and core glycosylation occur. Besides a role as a scaffold for binding SRP proteins (73), functional roles for SRP 7S RNA in elongation arrest and in interaction with the SRP receptor have been proposed (36,63,81) . Recently, it has b...

Alkaline extracellular protease (AEP) from Yarmwia /ipo/'ica is synthesized as a precursor with a 157 aa prepro-region. Signal peptide cleavage was shown to occur after Al a,, by N-terminal amino acid radiosequencing of the largest intracellular AEP precursor. AEP proteolytic activity was not required for AEP processing. After a change of the putative active site Ser to Ala, inactive AEP with the same mobility on SDS-PAGE as wild-type mature AEP was secreted. The role of dipeptidyl aminopeptidase (DPAPase) activity in AEP processing was also investigated. Mutations early in the -X-Ala-and -X-Pro-dipeptide stretch (Pro,, to Met which should prevent DPAPase processing and Al a,, to Val which should allow removal of only the first dipeptide) did not prevent synthesis of active mature AEP nor did use of the DPAPase inhibitor ProboroPro. Deletion of the entire dipeptide stretch (Ala,, to Pro, , ) resulted in intracellular accumulation of an AEP precursor, which surprisingly was not glycosylated, and little or no secretion of AEP-related polypeptides. Expression of AEP in wild-type and dpp1 dap2 Sacchammyces cerevisiae strains (lacking both the Golgi and vacuolar DPAPases) resulted in secretion of only mature AEP and no AEP precursors. Transit times and levels of AEP secretion were similar for both strains. These results indicate that the KEXZ-like cleavage after Lys, , , -Arg, , , , which yields mature active AEP can occur in the absence of DPAPase processing and that DPAPase processing is not necessary for secretion of mature active AEP.

Translocation of alkaline extracellular protease (AEP) into the endoplasmic reticulum of Yarrowia lipolytica is cotranslational and signal recognition particle (SRP)-dependent, whereas translocation of P17M AEP (proline to methionine at position 17, second amino acid in the pro-region) is posttranslational and SRP-independent. P17M signal peptide mutations that resulted in more rapid SRP-dependent translocation of AEP precursor were isolated. Most of these mutations significantly increased hydrophobicity, but the A12P/P17M mutation did not. The switch from SRP-dependent to SRP-independent translocation without a decrease in hydrophobicity (wild type to P17M) and restoration of SRPdependent translocation without an increase in hydrophobicity (P17M to A12P/P17M) indicate that some factor(s) in addition to hydrophobicity determines selection of targeting pathway. Models of extended forms of wild type and A12P/P17M signal peptides are kinked, whereas the P17M signal peptide is relatively straight. Possibly the conformation/orientation of signal peptides at the ribosomal surface affects SRP binding and consequently the targeting route to the endoplasmic reticulum. Kinked signal peptides might approach SRP more closely more often. Most likely, these effects were only detectable because of the short length and low average hydrophobicity of the AEP signal peptide.Whereas signal peptides have been extensively studied, the molecular details of their interactions with components involved in targeting, such as SRP, 1 are still unclear (Ref. 1, and for reviews, see Refs. 2 and 3). They are generally 15-30-amino acids long (for eukaryotes) and located at the N terminus. Signal peptides distinguish proteins that will enter the secretory pathway from those that will not. Although there is no sequence conservation between signal peptides, they have definite similarities and can be divided into regions. There usually is a net positive charge near the N-terminal end (n-region) followed by a hydrophobic stretch of amino acids (h-region) and a more polar C-terminal region usually containing the signal peptide cleavage site.Studies of mutated and synthetic signal peptides show that the degree of hydrophobicity affects the efficiency of translocation (for examples, see Refs. 4 -6). For carboxypeptidase Y from Saccharomyces cerevisiae (7,8), the wild type signal peptide did not interact in vitro with mammalian SRP, but a mutated form in which both glycines in the hydrophobic stretch were converted to leucines did. Recently, it was demonstrated that the average hydrophobicity of S. cerevisiae signal peptides is an important determinant of whether a protein is targeted by the SRP-dependent or -independent pathway (9). For proteins targeted by an SRP-independent pathway, the average hydrophobicity for 12 residues after the last positively charged residue of the n-region (HB12) is around 2.0 or less. For proteins targeted primarily by an SRP-dependent pathway, the HB12 values are significantly higher, around 3.0 or more.A kinetic partition...

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