Membrane adhesion dictates Golgi stacking and cisternal morphology

Abstract

Two classes of proteins that bind to each other and to Golgi membranes have been implicated in the adhesion of Golgi cisternae to each other to form their characteristic stacks: Golgi reassembly and stacking proteins 55 and 65 (GRASP55 and GRASP65) and Golgin of 45 kDa and Golgi matrix protein of 130 kDa. We report here that efficient stacking occurs in the absence of GRASP65/55 when either Golgin is overexpressed, as judged by quantitative electron microscopy. The Golgi stacks in these GRASP-deficient HeLa cells were normal both in morphology and in anterograde cargo transport. This suggests the simple hypothesis that the total amount of adhesive energy gluing cisternae dictates Golgi cisternal stacking, irrespective of which molecules mediate the adhesive process. In support of this hypothesis, we show that adding artificial adhesive energy between cisternae and mitochondria by dimerizing rapamycin-binding domain and FK506-binding protein domains that are attached to cisternal adhesive proteins allows mitochondria to invade the stack and even replace Golgi cisternae within a few hours. These results indicate that although Golgi stacking is a highly complicated process involving a large number of adhesive and regulatory proteins, the overriding principle of a Golgi stack assembly is likely to be quite simple. From this simplified perspective, we propose a model, based on cisternal adhesion and cisternal maturation as the two core principles, illustrating how the most ancient form of Golgi stacking might have occurred using only weak cisternal adhesive processes because of the differential between the rate of influx and outflux of membrane transport through the Golgi.

Keywords: GRASPs; tethers.

Fig. 1.

Double knockdown of GRASP65/55 or GM130/Golgin45 leads to significant disruption in Golgi cisternal flatness, but not Golgi disassembly. (A–C) Representative EM micrographs of HeLa cells treated with siRNAs against indicated adhesive proteins for 96 h. (Scale bar, 0.5 μm.) Control siRNA (A); GRASP65/55 siRNA (B); GM130/Golgin45 siRNA (C). Double knockdown of the two GRASPs or two Golgins resulted in significant disruption of Golgi cisternal flatness, but we did not observe significant Golgi unstacking under any double-knockdown conditions. For morphological criteria of Golgi unstacking, we looked for significant separation of cisternae from main body of the Golgi or significant vesiculation of the Golgi at the EM level. (D and E) K-S plots showing distribution of maximum cisternal luminal width in double-knockdown cells (D) or in single-knockdown cells (E), based on luminal width measurement using ImageJ software. (F) Table summarizing maximum luminal width measurement for both single- and double-knockdown cells. Results are expressed as the mean ± SEM. Numbers in parenthesis indicate the number of Golgi cisternae for which luminal width was measured. Note that all analysis passed the Student t test (P < 0.05), except for Golgin45-depleted cells. G, Golgi; n, nucleus; M, mitochondria; tER, transitional endoplasmic reticulum.

Fig. 2.

Disrupted Golgi cisternal flatness in the double-depletion cells can be reversed by rescue transfection. (A–D) K-S plots showing rescue of Golgi cisternal flatness by exogenous expression of the RNAi-resistant form of GRASP65 (A) or GRASP55 (B) in cells depleted of both GRASPs and by exogenous expression of the RNAi-resistant form of GM130 (C) or Golgin45 (D) in cells depleted of both Golgins. Representative electron micrographs of Golgi stacks in cells rescued with indicated cDNAs are shown in the right panel of each graph. (Scale bar, 0.5 μm.) (E) Table summarizing the results from rescue experiments. Results are expressed as the mean ± SEM. Numbers in parenthesis indicate the number of Golgi cisternae that luminal width was measured.

Fig. 3.

Morphologically and functionally normal Golgi stacks in GRASP65/55-deficient HeLa cells (A–C) K-S plots showing rescue of Golgi cisternal flatness by exogenous expression of Golgin45 (A) or GM130 (B) or Golgin97 as a control (C) in cells depleted of both GRASP65/55. Representative electron micrographs of Golgi stacks in cells rescued with indicated cDNAs are shown in the right panel of each graph. (Scale bar, 1 μm.) (D) Table summarizing the results from substitution experiments. Results are expressed as the mean ± SEM. Numbers in parenthesis indicate the number of Golgi cisternae that luminal width was measured.

Fig. 4.

Golgi–mitochondria hybrid stack formation driven by engineered ectopic adhesive interaction Representative electron micrographs from knock-sideways experiments. HeLa cells treated with siRNAs against GRASP65/55 for 72 h were rescued with GRASP55-FKBP-tagBFP for 24 h, followed by 3 h of treatment with Nocodazole (A); GRASP55-FKBP-tagBFP-rescued cells treated with AP21967 for indicated time to rapidly target the exogenous GRASP55 fusion protein onto mitochondria, which leads to Golgi-mitochondria hybrid stacks (B–D). G, Golgi; n, nucleus; M, mitochondria.

Fig. 5.

Illustrations describing the concept of the adhesion model and the simplest form of cisternal stacking that could occur via cisternal adhesion and Rab conversion as two fundamental principles of stacking mechanism. (A) Illustration explaining diverse morphologies of Golgi stacks based on the adhesion model: Golgi cisternae are analogous to a water-filled balloon that is densely covered with Velcro-like molecules (tethering proteins, GRASPs, or other adhesive proteins). At the low adhesive condition, two of these water-filled balloons (cisternae) would be barely adhered together. As the adhesive energy is progressively increased broadly across the surface of cisternae, the cisternae gets flattened as the cisternal adhesive strength overcomes the osmotic pressure within the cisternae. Examples of Golgi EM photos for progressively adhesive conditions are shown here. (Scale bar, 500 nm.) (B) Illustration depicting the simplest form of cisternal stacking, based on the adhesion model and Rab conversion. According to this model, cisternal stacking occurs because of significantly faster ER-to-Golgi anterograde cargo transport compared with cargo export from the trans-Golgi to the plasma membrane. Only weak tethering or adhesive processes may suffice if the rate of the ER-Golgi transport far exceeds that of Golgi–plasma membrane transport. Cisternal maturation initiates the stacking of newly forming cis-cisternae to now more distal cisternae.