The Sec14-superfamily and the regulatory interface between phospholipid metabolism and membrane trafficking

Abstract

A central principle of signal transduction is the appropriate control of the process so that relevant signals can be detected with fine spatial and temporal resolution. In the case of lipid-mediated signaling, organization and metabolism of specific lipid mediators is an important aspect of such control. Herein, we review the emerging evidence regarding the roles of Sec14-like phosphatidylinositol transfer proteins (PITPs) in the action of intracellular signaling networks; particularly as these relate to membrane trafficking. Finally, we explore developing ideas regarding how Sec14-like PITPs execute biological function. As Sec14-like proteins define a protein superfamily with diverse lipid (or lipophile) binding capabilities, it is likely these under-investigated proteins will be ultimately demonstrated as a ubiquitously important set of biological regulators whose functions influence a large territory in the signaling landscape of eukaryotic cells.

Figure 1. Crystal structure of an open Sec14p conformer

The Sec14p fold is comprised of twelve α-helices, six β-strands and eight 3₁₀ helices. The hydrophobic pocket is formed by six β-strands and three α-helices (helices α8, α9 and α11; in grey) and this pocket is predicted to be gated by the A₁₀/T₄ helix (in blue). The lipid binding pocket of apo-Sec14p is occupied by 2 molecules of β-octylglucoside (in yellow) and these molecules are oriented such that the acyl chains project into the pocket, disposing the headgroup towards solvent. The N-terminal α1, α2, α3 and α4 helices form the “tripod motif” that helps target Sec14p to Golgi membranes (in green). The “string motif” (in red) is comprised of a random coil regions and four 3₁₀ helices. This structural element wraps around the back of the lipid binding domain and is critical both for protein stability and the conformational dynamics that accompany the phospholipid exchange cycle.

Figure 2. The Sec14p pathway

‘Bypass Sec14p’ mutants suppress sec14 defects. This schematic illustrates the various effects of ‘bypass Sec14p’ mutants on phospholipid metabolism. The identification of lipid metabolic/binding activities in the ‘bypass Sec14p’ collection demonstrate Sec14p regulates Golgi secretory function via control of lipid metabolism/membrane lipid content. One ultimate recipient of these regulatory effects is posited to be the adenosine diphosphate-ribosylation factor (ARF) cycle. Putative downstream targets of ‘bypass Sec14p’ mutants include the ARF-GAPs Gcs1p and Age2p. All ‘bypass Sec14p’ mutants require an active phospholipase D (PLD) to exert this distinguishing phenotype. Mutations in the structural genes for choline kinase (CKI1) and choline phosphate cytidylytransferase (PCT1) inhibit PtdCho production and reduce consumption of DAG into PtdCho biosynthesis. DAG and PtdCho are posited to represent ‘pro-secretory’ and ‘anti-secretory’ lipids, respectively, on the basis of their opposing effects on the activity of the pro-secretory Gcs1p/Age2p ARFGAP activities. Genetic disruption of the SAC1 gene, which encodes a phosphoinositide phosphatase, results in accumulation of PtdIns-4-P. PtdIns-4-P is categorized as a pro-secretory phospholipid and its synthesis in the Golgi complex is primarily catalyzed by the PtdIns 4-OK kinase Pik1p. Accessory proteins that modulate PtdIns-4-P signaling are not well characterized. One candidate is Kes1p, an oxysterol binding protein homolog. Genetic data identify Kes1p as either an inhibitor of PtdIns-4-P synthesis, an activator of PtdIns-4-P degradation, or a binding protein that limits PtdIns-4-P accessibility for pro-secretory signaling. Thus, Kes1p is a negative regulator of the Sec14p pathway. That PtdIns-4-P also regulates Kes1p function is also plausible.

Figure 3. An intermembrane contact site model for SFH protein function

(A) SFH proteins (grey) may themselves be integral components of intermembrane contact sites, employing their intrinsic PtdIns-binding capacity (in black) to traffic PtdIns through a portal. (B) An SFH protein may employ its ability to present PtdIns to a phosphoinositide kinase to generate a phosphoinositide platform that recruits intrinsic component(s) (Factor X) of an intermembrane contact site. In this scenario, the SFH protein does not directly utilize its PtdIns-binding properties to impose PtdIns-trafficking specificity through such a site. Other lipid species could pass through such a site. This model most likely applies to the case of Sfh4p-dependent trafficking of PtdSer to an extramitochondrial PtdSer decarboxylase [; see text].

Figure 4. Sec14p like proteins in Arabidopsis thaliana

Bioinformatic analysis of the A. thaliana genome has identified at least 31 ORFs to encode proteins that share significant homology to Sec14p. Twelve ORFs consist of a Sec14p like domain positioned upstream of a nodulin domain whereas the remaining nineteen Sec14p like proteins consist of either a Sec14p lipid binding domain (LBD) alone or a Sec14-like LBD that precedes a GOLD domain.

Figure 5. AtSfh1p is required for root hair development in A. thaliana

Light microscopy of living, 10 day old, wild-type (left) and nullizygous Atsfh1^-/- seedlings (right). The defects in polarized membrane trafficking to the growing root tip in Atsfh1^-/- plants manifest themselves in the obvious short root hair phenotype of Atsfh1^-/- seedlings. Bars, 430μm.