Requirements for Transitional Endoplasmic Reticulum Site Structure and Function in Saccharomyces cerevisiae

Abstract

Secretory proteins are exported from the endoplasmic reticulum (ER) at specialized regions known as the transitional ER (tER). Coat protein complex II (COPII) proteins are enriched at tER sites, although the mechanisms underlying tER site assembly and maintenance are not understood. Here, we investigated the dynamic properties of tER sites in Saccharomyces cerevisiae and probed protein and lipid requirements for tER site structure and function. Thermosensitive sec12 and sec16 mutations caused a collapse of tER sites in a manner that depended on nascent secretory cargo. Continual fatty acid synthesis was required for ER export and for normal tER site structure, whereas inhibition of sterol and ceramide synthesis produced minor effects. An in vitro assay to monitor assembly of Sec23p-green fluorescent protein at tER sites was established to directly test requirements. tER sites remained active for approximately 10 min in vitro and depended on Sec12p function. Bulk phospholipids were also required for tER site structure and function in vitro, whereas depletion of phophatidylinositol selectively inhibited coat protein complex II (COPII) budding but not assembly of tER site structures. These results indicate that tER sites persist through relatively stringent treatments in which COPII budding was strongly inhibited. We propose that tER site structures are stable elements that are assembled on an underlying protein and lipid scaffold.