The authors have declared that no competing interests exist.
Analyzed the data: II AM. Wrote the paper: II AM. Conceived the study: II AM. Designed and ran simulations: II AM.
The generation of two non-identical membrane compartments via exchange of vesicles is considered to require two types of vesicles specified by distinct cytosolic coats that selectively recruit cargo, and two membrane-bound SNARE pairs that specify fusion and differ in their affinities for each type of vesicles. The mammalian Golgi complex is composed of 6–8 non-identical cisternae that undergo gradual maturation and replacement yet features only two SNARE pairs. We present a model that explains how distinct composition of Golgi cisternae can be generated with two and even a single SNARE pair and one vesicle coat. A decay of active SNARE concentration in aging cisternae provides the seed for a
We have developed a quantitative model to address a fundamental question in cell biology: How does the Golgi apparatus, an organelle composed of multiple cisternae that exchange vesicles, steadily maintains its inhomogeneous protein composition in the face of ongoing cisternal aging and replacement, and cargo entry and exit. We do not assume any
The Golgi apparatus is composed of multiple compartments, called cisternae, typically 6–8 in mammalian cells. The individual cisternae are enriched in glycosylation and other enzymes, which form distinct but overlapping gradients with peaks in the
As anterograde cargo traverses the Golgi apparatus from
A) ER-derived vesicles (beige) fuse with each other to yield the first, most
It has been shown that Golgi resident proteins shuttle between the cisternae in vesicles
Glick et al. provided one piece of explanation with a simple model according to which competition of Golgi proteins for incorporation into retrograde-destined vesicles accounts for their sorting within the Golgi cisternae
Fusion of vesicles with acceptor membranes is specified by
We present a model of inter-cisternal vesicular transport in which we do not assume any
We assume that
The Golgi consists of a stack of
Along with cisternal progression, vesicles containing SNAREs and Golgi enzymes continuously bud from each cisterna. We assume that the vesicles provide local transport and can only fuse with the neighboring
SNAREs and Golgi enzymes are uploaded into a vesicle via competitive binding to a fixed number of vesicular sites. We assume that the vesicular transport results primarily in the movement of cargo without any significant change in the volume and budding surface area of the cisternae. This is supported by observations that the size of all cisternae is similar
Functioning of the model hinges on two general principles: Establishment and maintenance of a directed retrograde vesicular flux and sorting of the vesicular cargo via competition for binding sites.
To reveal the universality of the proposed self-establishing mechanism of vesicular traffic directionality we first consider the simplest possible setup, a single cognate SNARE pair and vesicle type. We assume that the rate of vesicular fusion is proportional to the product of the concentrations of the SNAREs present in vesicles and cisternae, respectively. The precise nature of SNARE molecules does not have to be specified here. We can even consider the SNAREs as mere proxy for fusion-specifying factors. The probability for a vesicle to fuse with a given cisterna depends solely on the cisternal concentration of compatible SNAREs, and cisternae with higher SNARE concentration have a higher probability to absorb vesicles. A retrograde vesicular flux thus requires a
We propose that key to a robust
The “seed” SNARE gradient generated in this manner sets a preference for vesicles to fuse with
We note that at steady state the vesicular flux does not depend on the concentration of SNAREs in the vesicles: Lower concentrations of vesicular SNAREs are compensated by a higher steady state number of vesicles. Naturally, a vesicle should contain a minimum number of vesicular SNARE molecules to ensure any fusion at all.
The calculation of the steady state SNARE gradient and vesicular flow are presented in the
Next, we investigated how retrograde vesicular flow, created by the cisternal SNARE gradient, maintains the inhomogeneous steady state distribution of Golgi enzymes during cisternal maturation. To this end, we further developed the principle proposed by Glick et al. that attributes the different cisternal enzyme profiles to the competition of enzymes for the binding sites in vesicles
We find that the distribution of enzymes radically depends on whether vesicles originating from the first cisterna can exit the Golgi and fuse with its
On the other hand, if none of the enzyme-carrying vesicles can escape to the ER, the cisternal distribution of all enzymes converges onto a single peak form (
We also observe that, as discussed in
The quantitative details of the calculation of the steady state enzyme concentrations, including
We now apply the general mechanisms of fusion asymmetry and competitive vesicle binding to explain the specific SNARE and enzyme distributions as they are actually observed in the mammalian Golgi. The important adjustment to our basic model is that the Golgi apparatus features not one, but two cognate SNARE pairs. The first pair, which we label
To reproduce three Golgi enzyme peaks in concurrence with the experimentally observed distributions of the
In the Golgi, only the
In addition to the two Golgi SNAREs, we consider a third v-SNARE, which mediates the fusion of Golgi-derived vesicles with the ER. It is ERS24, which thus has a dual function as part of a t-SNARE complex in intra Golgi transport and as v-SNARE in Golgi-to-ER transport. The corresponding ER t-SNARE does not leave the ER and is therefore not considered here
Apart from the SNARE specifications, we implemented a similar set of minimal assumptions as for the single SNARE scenario:
The rate of vesicular fusion with Golgi cisternae is determined by both
All SNAREs and Golgi enzymes compete for the same binding sites in the vesicles. This is in agreement with the findings for ER-derived COPII vesicles, the only instance where cargo-competition for coat binding has been elucidated to date
However, our model reproduces the enzyme segregation as well in the case when the enzymes compete only with each other and not with SNAREs for vesicular binding sites, as shown in
Vesicles fuse locally, i.e. with the
The age-dependent decrease (loss) of the cisternal concentration of the
We found a good qualitative agreement between our results and the experimentally observed concentration profiles. With the proper choice of dissociation constants (
Substance | Dissociation constant |
ER v-SNARE | 0.2 |
0.4 | |
0.4 | |
1 | |
5 | |
1.4 | |
Medial enzyme | 2.5 |
5 |
Our prediction that
Based on the SNARE dissociation constants that yielded the experimentally observed protein gradients (
So far we assumed that vesicles only fuse with the immediate neighbors of their progenitor cisternae. A stacked Golgi, however, is not a requirement for Golgi asymmetry and cisternal maturation, which are also observed in
We suggest therefore, that a realistic description of fusion probability in
Golgi enzyme concentrations are normalized by their maximum value. The parameters are: Decay rates for the
We present a simple model that explains the establishment and maintenance of directed vesicular flow and concentration gradients in the Golgi apparatus, an organelle system that undergoes constant rejuvenation by adding a new cisterna at the cargo-entering
The “seeding” temporal decay of cisternal SNARE concentrations occurs via several mechanisms: i) Retrieval to the ER alone can account for the loss of the SNAREs present mostly in the young cisternae. However, the retrieval to the ER of the Golgi SNAREs from the medial and
Once the retrograde vesicular flux is established, different affinities of Golgi enzymes for the vesicles explain the enzyme peaks in
Our simulations are insensitive to a broad spectrum of initial conditions. Regardless of whether we started with a single cisterna and added new cisternae one by one as it would occur during Golgi
An important question is the relevance and specificity of constants used for the modeling. Naturally, the range of admissible constants narrows as one reproduces more detailed and specific scenarios. Our first observation, that a temporal loss of SNAREs results in directed vesicular flux, is very general and holds for virtually any set of constants (see
We observed that the distinct enzyme peaks can be achieved with just one cognate SNARE pair. Why then does the Golgi afford two SNARE pairs? One proposal, put forward by Volchuk et al., is that only the
According to our model, the experimentally observed steep
In summary, we have presented an explanation for why the minimal requirement of one SNARE pair and one vesicle type for the generation and maintenance of each distinct organelle
Here we do not specify the nature of t- and v- SNAREs, simply calling fusiogenic molecules present in a vesicle and cisterna v-SNAREs and t-SNAREs. The chemical distinction between t- and v-SNAREs will be stated later. However, to keep the same notations throughout the paper, we use the specific
The number of vesicles that bud from the
A vesicle emitted from the
The rate equation that describes the evolution of the t- SNARE concentration in the
To complete the description of t-SNARE distribution, the vesicular transport
We measure cisternal concentrations of SNAREs and other molecules in the units of their initial concentrations in the first cisterna and the natural unit of time is the period of cisternal maturation
This cisternal maturation scenario together with
Consider a hypothetical system where the number of cisternae is non-biologically large. For older cisternae, the concentrations of SNAREs are small,
We observe that in the asymptotic regime, the steady state t-SNARE concentration decays exponentially with the number of cisterna,
The steady state gradient has the exponentially decaying form,
The necessity of the loss term for establishing the gradient by breaking the initial symmetry between the cisternae is clearly revealed by the following analytic argument: For a small loss rate (
The transport of Golgi enzymes with cisternal concentrations
When the retrograde vesicular transport is counterbalanced by the anterograde cisternal progression, the enzyme distribution reaches its steady state. The nature of the steady state depends on the boundary conditions imposed on the
Steady state distribution of the same enzymes as in
Putting together the two mechanisms considered above, we introduce a realistic model of Golgi transport. It describes the evolution of 8 distinct types of molecules:
The escape of a fraction of vesicles from the Golgi to the ER provides one part of a loss mechanism necessary for seeding the gradient of t-SNAREs. Yet we do not exclude the possibility of other mechanisms of t-SNARE decay, so the
To model the vesicular transport in yeast, we used an equation similar to
We acknowledge the contribution of T. Rapoport who suggested this study and critically commented on the manuscript. A.M. also thanks her lab for helpful discussions.