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. 1998 Sep 15;95(19):11175-80.
doi: 10.1073/pnas.95.19.11175.

Endoplasmic reticulum membrane localization of Rce1p and Ste24p, yeast proteases involved in carboxyl-terminal CAAX protein processing and amino-terminal a-factor cleavage

W K Schmidt  1 , A Tam, K Fujimura-Kamada, S Michaelis
Affiliations

Endoplasmic reticulum membrane localization of Rce1p and Ste24p, yeast proteases involved in carboxyl-terminal CAAX protein processing and amino-terminal a-factor cleavage

W K Schmidt et al. Proc Natl Acad Sci U S A. .

Abstract

Proteins terminating in the CAAX motif, for example Ras and the yeast a-factor mating pheromone, are prenylated, trimmed of their last three amino acids, and carboxyl-methylated. The enzymes that mediate these activities, collectively referred to as CAAX processing components, have been identified genetically in Saccharomyces cerevisiae. Whereas the Ram1p/Ram2p prenyltransferase is a cytosolic soluble enzyme, sequence analysis predicts that the other CAAX processing components, the Rce1p and Ste24p proteases and the Ste14p methyltransferase, contain multiple membrane spans. To determine the intracellular site(s) at which CAAX processing occurs, we have examined the localization of the CAAX proteases Rce1p and Ste24p by subcellular fractionation and indirect immunofluorescence. We find that both of these proteases are associated with the endoplasmic reticulum (ER) membrane. In addition to having a role in CAAX processing, the Ste24p protease catalyzes the first of two cleavage steps that remove the amino-terminal extension from the a-factor precursor, suggesting that the first amino-terminal processing step of a-factor maturation also occurs at the ER membrane. The ER localization of Ste24p is consistent with the presence of a carboxyl-terminal dilysine ER retrieval motif, although we find that mutation of this motif does not result in mislocalization of Ste24p. Because the ER is not the ultimate destination for a-factor or most CAAX proteins, our results imply that a mechanism must exist for the intracellular routing of CAAX proteins from the ER membrane to other cellular sites.

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Figures

Figure 1
Figure 1
The predicted transmembrane structure and motifs of Rce1p and Ste24p. Kyte and Doolittle hydropathy plots of Rce1p and Ste24p (Lower) are rendered as schematic models (Upper). Predicted membrane spans are indicated [filled bars (Upper) and filled peaks (Lower)], as are the characteristic metalloprotease motif (HEIGH) and ER retrieval motif (KKKN) of Ste24p. At the top of each diagram and along the x-axis of the hydropathy plots, a scale for amino acid position is shown.
Figure 2
Figure 2
Complementation of the CAAX protease-deficient Δrce1 Δste24 mutant by RCE1∷HA and HA∷STE24 and immunodetection of the epitope-tagged proteins. (A) Strains were tested for their mating efficiency under stringent conditions by a patch mating assay, with the SM1068 (MATα lys1) mating partner, as described (11). The growth of prototrophic diploids is indicative of mating. MATa strains tested are: SM3614, SM1058/pRS316, SM3614/pSM1093, SM3614/pSM1107, SM3614/pSM1275, SM3614/pSM1314 for a-f), respectively. (B) Blots were probed with the anti-HA antibody (lanes 1–4) or the Rce1p antibody (lanes 5–8), as described in Materials and Methods. The approximate molecular mass (kDa) of a set of protein standards is indicated for each blot. Strains used are: SM3614/pRS316, SM3614/pSM1107, SM3614/pSM1314, SM1058/pSM1097 and pSM1314, SM3613, SM1058/pRS314, SM3613/pSM1275, and SM1058/pHY01 for lanes 1–8, respectively.
Figure 3
Figure 3
Distribution of CAAX proteases and associated AAXing activity by subcellular fractionation. (A) A total yeast lysate derived from a strain expressing HA-Ste24p and Kex2p-HA was fractionated on a sucrose step gradient. Equivalent volumes from each fraction (50 μl) were solubilized in Laemmli’s sample buffer, subjected to SDS/15% PAGE, transferred to nitrocellulose, and probed with antibodies for Rce1p, HA (HA-Ste24p and Kex2p-HA), and organellar markers. Gradient fractions were also assayed for protein concentration and AAXing activity, as described in Materials and Methods. The broken line represents an arbitrary division between light and heavy membranes. The strain used is SM3365/pSM962/pSN218/pSM1153. (B) Total membranes prepared from yeast cells of the indicated genotypes were assayed for AAXing activity, as above. Strains used are (left to right) SM1058/pRS316, SM3103/pRS316, SM3613/pRS316, and SM3614/pRS316.
Figure 4
Figure 4
Immunofluorescence localization of Rce1p-HA and HA-Ste24p to the ER compartment. Indirect immunofluorescence detection of proteins was performed to examine the localization of CAAX components in relation to ER (Kar2p), plasma membrane (Pma1p), and Golgi (Och1p-HA) markers. Coimmunofluorescence was carried out by using the anti-HA antibody (A–E, Left), the anti-Kar2p antibody (A, C and D, Right) or the anti-Pma1p antibody (B, Right). Nonspecific staining for the anti-HA antibody is shown in F. Primary and appropriate fluorescent secondary antibodies were used at empirically determined dilutions. Images were captured at ×100 magnification by using a Zeiss microscope equipped with fluorescence optics and a charge-coupled device camera. Fluorescence filter sets were optimized such that no bleed-through was observed between fluorescent channels. Strains used are A–B: SM1058/pSM1314; C–F: SM3060/pSM1107, SM3103/pSM1291, SM1058/pOH and SM1058, respectively.
Figure 5
Figure 5
Model for the organization of a-factor biogenesis components. The a-factor precursor is modified by several components. Isoprenylation is carried out by the cytosolic Ram1p/Ram2p complex. The Rce1p and Ste24p proteases and the Ste14p methyltransferase are localized to the ER membrane, presumably with cytosolically disposed active sites. Axl1p is localized to a different, as yet unidentified, compartment. The Ste6p transporter is localized to the plasma membrane. Routing through these processing stations is required for the maturation of the a-factor precursor and for the export of mature a-factor.
Figure 6
Figure 6
The biogenesis of CAAX proteins requires routing from the ER membrane. Whereas CAAX processing occurs at the ER membrane, CAAX proteins are destined to other membrane sites, for instance the plasma membrane. Transport of CAAX proteins such as Ras from the ER membrane to the plasma membrane could involve trafficking by one of the three possible mechanisms shown: (1) diffusional translocation across the cytoplasm, (2) transport along the cytoplasmic face of known organelles such as those of the secretory pathway, or (3) transport via an as yet unidentified protein, lipid, or vesicular carrier.
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References

    1. Clarke S. Annu Rev Biochem. 1992;61:355–386. - PubMed
    1. Zhang F L, Casey P J. Annu Rev Biochem. 1996;65:241–269. - PubMed
    1. Chen P, Sapperstein S K, Choi J C, Michaelis S. J Cell Biol. 1997;136:251–269. - PMC - PubMed
    1. McGrath J P, Varshavsky A. Nature (London) 1989;340:400–404. - PubMed
    1. Schafer W, Trueblood C, Yang C, Mayer M, Rosenberg S, Poulter C, Kim S-H, Rine J. Science. 1990;249:1133–1139. - PubMed

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