ER exit sites (ERES) and ER-mitochondria encounter structures (ERMES) often localize proximally.

To understand the potential interplay between vesicular trafficking and direct membrane contact sites-mediated transport, we selected the endoplasmic reticulum (ER), which participates in both modes of inter-organelle transport. ER-mitochondria encounter structures (ERMES) are direct membrane contact junctions that mediate macromolecule exchange, while the secretory pathway originates at ER exit sites (ERES). Using the budding yeast Pichia pastoris, we documented that ERMES resident proteins are often juxtaposed with ERES markers. We further demonstrated that ERES form de novo almost always near a pre-existing ERMES. Disruption of either ERES or ERMES affects the other. Djp1, a chaperone reported to mediate mitochondrial import of ER-resident proteins, localizes at the ERES-ERMES proximal region. Our results indicate a potential functional link between ERES-ERMES proximity and mitochondrial protein import.

Exofacial membrane composition and lipid metabolism regulates plasma membrane P4-ATPase substrate specificity.

The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. Regulation of membrane asymmetry is a fundamental characteristic of membrane biology and is crucial for signal transduction, vesicle transport, and cell division. The type IV family of P-ATPases, or P4-ATPases, establishes membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is unclear how P4-ATPases sort through the spectrum of membrane lipids to identify their desired substrate(s) and how the membrane environment modulates this activity. Therefore, we tested how the yeast plasma membrane P4-ATPase, Dnf2, responds to changes in membrane composition induced by perturbation of endogenous lipid biosynthetic pathways or exogenous application of lipid. The primary substrates of Dnf2 are glucosylceramide (GlcCer) and phosphatidylcholine (PC, or their lyso-lipid derivatives), and we find that these substrates compete with each other for transport. Acutely inhibiting sphingolipid synthesis using myriocin attenuates transport of exogenously applied GlcCer without perturbing PC transport. Deletion of genes controlling later steps of glycosphingolipid production also perturb GlcCer transport to a greater extent than PC transport. In contrast, perturbation of ergosterol biosynthesis reduces PC and GlcCer transport equivalently. Surprisingly, application of lipids that are poor transport substrates differentially affects PC and GlcCer transport by Dnf2, thus altering substrate preference. Our data indicate that Dnf2 exhibits exquisite sensitivity to the membrane composition, thus providing feedback onto the function of the P4-ATPases.

ER arrival sites associate with ER exit sites to create bidirectional transport portals.

COPI vesicles mediate Golgi-to-ER recycling, but COPI vesicle arrival sites at the ER have been poorly defined. We explored this issue using the yeast Pichia pastoris. ER arrival sites (ERAS) can be visualized by labeling COPI vesicle tethers such as Tip20. Our results place ERAS at the periphery of COPII-labeled ER export sites (ERES). The dynamics of ERES and ERAS are indistinguishable, indicating that these structures are tightly coupled. Displacement or degradation of Tip20 does not alter ERES organization, whereas displacement or degradation of either COPII or COPI components disrupts ERAS organization. We infer that Golgi compartments form at ERES and then produce COPI vesicles to generate ERAS. As a result, ERES and ERAS are functionally linked to create bidirectional transport portals at the ER-Golgi interface. COPI vesicles likely become tethered while they bud, thereby promoting efficient retrograde transport. In mammalian cells, the Tip20 homologue RINT1 associates with ERES, indicating possible conservation of the link between ERES and ERAS.

The golgin PpImh1 mediates reversible cisternal stacking in the Golgi of the budding yeast Pichia pastoris.

The adhesive force for cisternal stacking of Golgi needs to be reversible - to be initiated and undone in a continuous cycle to keep up with the cisternal maturation. Microscopic evidence in support of such a reversible nature of stacking, in the form of 'TGN peeling,' has been reported in various species, suggesting a potential evolutionarily conserved mechanism. However, knowledge of such mechanism has remained sketchy. Here, we have explored this issue in the budding yeast Pichia pastoris which harbors stacked Golgi. We observed that deletion of GRIP domain golgin P. pastoris (Pp)IMH1 increases the peeling of late cisterna, causing unstacking of the Golgi stack. Our results suggest that the PpImh1 dimer mediates reversible stacking through a continuous association-dissociation cycle of its GRIP domain to the middle and late Golgi cisterna under the GTP hydrolysis-based regulation of Arl3-Arl1 GTPase cascade switch. The reversible cisternal stacking function of PpImh1 is independent of its vesicle-capturing function. Since GRIP domain proteins are conserved in plants, animals and fungi, it is plausible that this reversible mechanism of Golgi stacking is evolutionarily conserved.This article has an associated First Person interview with the first author of the paper.

Vps74p controls Golgi size in an Arf1-dependent manner.

The oncogene GOLPH3 is implicated in Golgi size regulation, a function yet to be experimentally linked to its PI4P effector function or the Golgi cisternal maturation in general. Moreover, its yeast homolog, Vps74p is not yet implicated in Golgi size regulation. Our results indicate that VPS74 deletion increases the late Golgi cisternal size and the cisternal maturation frequencies, and destabilizes the Golgi PI4P gradient in budding yeast. Overexpression of Arf1 can suppress this cisternal enlargement and increased maturation frequency phenotype of ∆vps74. ∆arf1 alters Vps74p and PI4P distribution along the Golgi stacks. We conclude that Vps74p, the downstream effector of Arf1, regulates Golgi size by altering its cisternal maturation frequency and by maintaining the PI4P distribution along the Golgi compartments.

Identification and characterization of GRIP domain Golgin PpImh1 from Pichia pastoris.

Budding yeast Pichia pastoris has highly advanced secretory pathways resembling mammalian systems, an advantage that makes it a suitable model system to study vesicular trafficking. Golgins are large Golgi-resident proteins, primarily reported to play role in cargo vesicle capture, but details of such mechanisms are yet to be deciphered. Golgins that localize to the Golgi via their GRIP domain, a C-terminal Golgi anchoring domain, are known as GRIP domain Golgins. In this present study, we have identified and functionally characterized a homologue of one such GRIP domain Golgin protein, Imh1, from the budding yeast P. pastoris. We have demonstrated that the GRIP domain present at the C-terminal of P. pastoris Imh1 (PpImh1) functions as its Golgi-targeting sequence. Using a combination of yeast two-hybrid analysis, dynamic light scattering and electron microscopy, we have shown that PpImh1 can self-associate and form a homodimer. Analysis of purified recombinant PpImh1 by CD spectroscopy indicates the presence of an 85% α-helical structure, a characteristic of high-content α-helical coiled-coil sequences normally present in other Golgin family proteins. Two-hybrid analysis indicated self-interaction between C-terminal fragments, yet N-terminal fragments do not mediate any such form of self-interaction, suggesting that PpImh1 may form a parallel dimer. Electron microscopy data indicates that PpImh1 forms extended rod-like homo-dimeric molecules with splayed N-terminal end which can act as a tether for capturing vesicles. Our study provides the first evidence in support of the dimeric Y-shaped structure for any Golgin in the budding yeast.

Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation.

Regulation of the size and abundance of membrane compartments is a fundamental cellular activity. In Saccharomyces cerevisiae, disruption of the ADP-ribosylation factor 1 (ARF1) gene yields larger and fewer Golgi cisternae by partially depleting the Arf GTPase. We observed a similar phenotype with a thermosensitive mutation in Nmt1, which myristoylates and activates Arf. Therefore, partial depletion of Arf is a convenient tool for dissecting mechanisms that regulate Golgi structure. We found that in arf1Δ cells, late Golgi structure is particularly abnormal, with the number of late Golgi cisternae being severely reduced. This effect can be explained by selective changes in cisternal maturation kinetics. The arf1Δ mutation causes early Golgi cisternae to mature more slowly and less frequently, but does not alter the maturation of late Golgi cisternae. These changes quantitatively explain why late Golgi cisternae are fewer in number and correspondingly larger. With a stacked Golgi, similar changes in maturation kinetics could be used by the cell to modulate the number of cisternae per stack. Thus, the rates of processes that transform a maturing compartment can determine compartmental size and copy number.