Sec14 regulation of phospholipid metabolism. A, regulation of phospholipid metabolism by Sec14 is thought to be lipid ligand-dependent. Sec14 bound to PC regulates PC metabolism, whereas Sec14 bound to PI controls phosphoinositide metabolism. PC-bound Sec14 is thought to inhibit PC synthesis at the rate-limiting step of Pct1 while at the same time inhibiting PC turnover by the phospholipase D Spo14 (to decrease the production of choline to prevent supply of PC precursor). At the same time Sec14-PC shunts PC turnover toward the phospholipase B Nte1, away from supplying choline for PC synthesis. In this way, Sec14 acts as a homeostat for regulation of PC levels. Sec14 bound to PI maintains PI-4P levels, although whether this is by activating the PI 4-kinase Pik1 is not known. The solid arrows indicate metabolic pathways; the dotted arrows activation, and the dotted lines inhibition. GPC, glycerophosphocholine. B, pathways in and out of the trans-Golgi. A subset of vesicles destined to fuse with the trans-Golgi do so via the GARP complex (contains Vps51, Vps52, Vps53, and Vps54). The GARP complex binds Ypt6, a Rab regulated by the GEF pair Ric1/Rgp1. The GARP complex also directly interacts with the Arf-like GTPase Arl1 that is anchored to the trans-Golgi via Arl3 and Sys1. Fusion to the trans-Golgi is mediated in part by the t-SNARE Tlg2 and its accessory protein Vps45. A second Golgi-associated complex, the transport protein particle II (TRAPP II) complex (contains the essential subunits Bet3, Bet5, Trs20, Trs23, Trs31, Trs120, and Trs130 and the nonessential subunits Trs33, Trs65, and Trs85), acts as a GEF for the Rab pair Ypt31/32 and mediates transport both in and out of the trans-Golgi.

Genes whose inactivation aggravates growth of sec14^ts cells. A, pie chart of the functions of the 40 genes identified by the SGA screen whose inactivation decreased growth of sec14^ts cells. A subset of these genes (SPO14, YPT31, TRS33, TLG2, GYP1, and VPS1) had been identified previously as genetic interactors in cells with decreased Sec14 function (2). B, isogenic mutant strains with the indicated genotypes were grown to mid-log phase in synthetic complete medium at 25 °C; identical numbers of cells were serial diluted, spotted onto agar plates, and grown for 3 days at the indicated temperatures. Invertase secretion is the ratio of external invertase enzyme activity to total (internal + external) invertase activity. Invertase data are the mean of three individual experiments performed in triplicate. Standard errors of the mean are indicated in the figure.

Cells with reduced Sec14 function do not have a major defect in bulk protein secretion. A, mid-log phase cells were incubated at 25 °C or shifted to 37 °C for 1 h. Cells were harvested and resuspended in 10 mm NaN₃, 10 mm KF and incubated on ice. Cells were washed and resuspended in buffer containing 1.4 m sorbitol and zymolyase. Resulting spheroplasts were pelleted and proteins separated by SDS-PAGE. Bgl2 was detected by Western blot. B, wild type, sec14^ts, and sec18^ts cells (strains used were BY4741 (wild type), CMY505 (sec14^ts), X2180 (wild type), SF292-1A (sec14^ts), and SF282-1D (sec18^ts)) were grown to mid-log phase at 25 °C, shifted to 37 °C for 10 min, incubated with [³⁵S]methionine/cysteine for 10 min, and then with 10 mm unlabeled methionine/cysteine for 15 and 30 min at 37 °C. Proteins in the medium were separated by SDS-PAGE, and the gel was exposed to x-ray film.

Sec14 regulates Snc1 cycling. A, wild type and sec14^ts cells expressing GFP-Snc1 were grown to early log phase at 25 °C; a portion was shifted to 37 °C, and live cells were imaged using DIC and fluorescence microscopy at the indicated time points. B, wild type SEC14 gene was replaced with the sec14^ts allele in yeast strains where RFP was fused to the organellar markers Chc1 (trans-Golgi) and Snf7 (endosomes). These strains were transformed with the plasmid expressing GFP-Snc1, and colocalization of GFP-Snc1 was determined in sec14^ts cells after cells were shifted to 37 °C for 30 min. C, wild type and sec14^ts cells (strains BY4741 and CMY505) were shifted to 37 °C for 30 min resulting in GFP-Snc1 localization to the trans-Golgi in sec14^ts cells. Lat-A was added for 10 min to prevent GFP-Snc1 cycling back into the cell from the plasma membrane, and cells were grown for a further 15-30 min at 37 °C. Live cells were visualized using DIC and fluorescence microscopy. The number of cells from random fields (four fields from two separate experiments totaling at least 100 cells) was visually assessed for GFP-Snc1 at the plasma membrane using a double-blinded protocol.

Sec14 is required for trafficking of endosomes to the vacuole. A, wild type and sec14^ts MAT α cells (BY4742 and CMY506) were grown to mid-log phase at 25 °C and shifted to 37 °C for 2 h. Proteins were extracted, separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with antibodies for Ste3 and Pgk1. B, wild type (BY4741) and sec14^ts (CMY505) cells were grown to mid-logarithmic phase at 25 °C, shifted to 37 °C for 15 min, labeled for 2 min with 40 μm FM4-64, and incubated post-label in fresh pre-warmed 37 °C medium for the indicated times. Live cells were visualized using DIC and fluorescence microscopy. C, FM4-64 was colocalized with the endosome marker Vps27-GFP at various time points after the shift to 37 °C.

Tethering complex defects aggravate Sec14 function. Isogenic mutant strains with the indicated genotypes were grown to mid-log phase in synthetic complete medium at 25 °C, and identical numbers of cells were serial diluted, spotted onto agar plates, and grown for 3 days at the indicated temperatures.

Regulation of specific vesicular transport processes based on Sec14 lipid ligand occupancy. A, isogenic mutant strains with the indicated genotypes transformed with low copy plasmids expressing Sec14, Sec14-PC, or a high copy plasmid expressing Sfh4 were grown to mid-log phase in synthetic complete medium at 25 °C, and identical numbers of cells were serial diluted, spotted onto agar plates, and grown for 3 days at the indicated temperatures. B, tabulation of the ability of Sec14, Sec14-PC, and Sfh4 to rescue cell growth of sec14^ts cells with the indicated inactivated genes.

Regulation of vesicular transport by Sec14. A, Sec14 facilitates nonendosomal and endosomal transport pathways from the trans-Golgi to the plasma membrane (pink and blue dotted lines). At least one other route from the trans-Golgi to the plasma membrane is not facilitated by Sec14. Transport from the plasma membrane to both the trans-Golgi and vacuole via endosomes is also regulated by Sec14. B, genetic evidence suggest Sec14 regulates endosome trafficking to the trans-Golgi via all major routes into this organelle by regulating Golgi PI-4P levels. Many of the gene deletions that aggravate growth of sec14^ts cells also aggravate growth of cells containing a temperature-sensitive allele of the Golgi PI 4-kinase, PIK1; Golgi PI-4P levels are decreased in sec14^ts and pik1^ts cells (2). This implies that regulation of Golgi PI-4P levels by Sec14 is necessary for regulation of import and export of vesicles from the trans-Golgi. The processes affected could include GARP complex (Vps51-54) binding to the t-SNARE Tlg2 that, together with Vps45, aid in targeting and fusion of endosome-derived vesicles with the trans-Golgi. Fusion is promoted by interaction of the GARP complex with the Rab GTPase Ypt6 that is in turn activated by the heterodimeric GEF Ric1/Rgp1. GARP also binds the Arf-like GTPase Arl that is localized to the Golgi by a second Arf-like GTPase Arl3 that interacts with the Golgi resident protein Sys1. The function of the Rab GTPase Ypt31 appears to be to regulate trafficking both in and out of the Golgi. Ypt31 is activated by the 10-subunit TRAPP II complex of which Trs33. Trs65, and Trs85 are nonessential. Reduction of Sec14 function results in aberrant Golgi PI-4P levels directly confounding vesicle import and export. The defects in endosome trafficking from the plasma membrane to the vacuole could be due to improper endosome trafficking because of miscommunication between Rab cascades into and out of the trans-Golgi.