RESUMEN
Regulation of organelle size and shape is a poorly understood but fascinating subject. Several theoretical studies were reported on Golgi size regulation, but a combination of experimental and theoretical approaches is rare. In combination with the quantitative microscopy and a coarse-grained simulation model, we have developed a technique to gain insights into the functions of potential regulators of Golgi size in budding yeast Saccharomyces cerevisiae. To validate our method, we tested wild-type and arf1[Formula: see text] strain harboring early and late Golgi cisternae labeled with green and red fluorescent fusions. Our concentration-dependent maturation model prediction concurs with most of the experimental results for both wild-type and arf1[Formula: see text] strains. Decisive match of simulation and experimental data provide insight into such specific factor's function in regulating the Golgi size. Details of the complex multifactorial network of Golgi size regulation can be deciphered in the future using a similar combination of quantitative microscopy and in silico model.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Simulación por Computador , Aparato de Golgi/metabolismo , Microscopía , Modelos Biológicos , Tamaño de los Orgánulos , Saccharomyces cerevisiae/citologíaRESUMEN
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.
Asunto(s)
Factor 1 de Ribosilacion-ADP/genética , Regulación Fúngica de la Expresión Génica , Aparato de Golgi/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factor 1 de Ribosilacion-ADP/deficiencia , Transporte Biológico , Aparato de Golgi/ultraestructura , Mutación , Ácidos Mirísticos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
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.